Water soluble texaphyrin metal complex preparation

ABSTRACT

The present invention involves water soluble hydroxy-substituted texaphyrins retaining lipophilicity, the synthesis of such compounds and their uses. These expanded porphyrin-like macrocycles are efficient chelators of divalent and trivalent metal ions. Various metal (e.g., transition, main group, and lanthanide) complexes of the hydroxy-substituted texaphyrin derivatives of the present invention have unusual water solubility and stability. They absorb light strongly in a physiologically important region (i.e. 690-880 nm). They have enhanced relaxivity and therefore are useful in magnetic resonance imaging. They form long-lived triplet states in high yield and act as photosensitizers for the generation of singlet oxygen. Thus, they are useful for inactivation or destruction of enveloped viruses, mononuclear or other cells infected with such viruses as well as tumor cells. They are water soluble, yet they retain sufficient lipophilicity so as to have greater affinity for lipid rich areas such as atheroma and tumors. They may be used for magnetic resonance imaging followed by photodynamic tumor therapy in the treatment of atheroma and tumors. These properties, coupled with their high chemical stability and appreciable solubility in water, add to their usefulness.

The government has certain rights in the present invention pursuant toNational Institutes of Health contract AI28845.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of Ser. No. 07/822,964,filed Jan. 21, 1992 now U.S. Pat. No. 5,252,720. Ser. No. 07/822,964 isa continuation-in-part of U.S. Ser. No. 771,393 filed Sep. 30, 1991 nowabandoned which is a continuation-in part of U.S. Ser. No. 539,975 filedJun. 18, 1990 (since issued as U.S. Pat. No. 5,162,509, Nov. 10, 1992)which is a division of U.S. Ser. No. 320,293 filed Mar. 6, 1989 (sinceissued as U.S. Pat. No. 4,935,498, Jun. 19, 1990)and is a continuationof international application no. PCT/US90/01208, internationally filed 6March, 1990, all of which are incorporated by reference herein.

BACKGROUND OF THE INVENTION

The porphyrins and related tetrapyrrole macrocycles are among the mostversatile of tetradentate ligands¹. Attempts to stabilize highercoordination geometries with larger porphyrin-like aromatic macrocycleshave met with little success.²⁻¹³ Only the uranyl complex of"superphthalocyanine" has been isolated and characterized structurally,²although several other large porphyrin-like aromatic macrocycles,including the "sapphyrins",³,6 "oxosapphyrins",⁶,7 "platyrins",⁸"pentaphyrin",⁹ and "[26]porphyrin",¹⁰ have been prepared in their metalfree forms. Large, or "expanded" porphyrin-like systems are of interestfor several reasons: They could serve as aromatic analogues of thebetter studied porphyrins²⁻¹⁰ or serve as biomimetic models for these orother naturally occurring pyrrole-containing systems.³⁶,13a In addition,large pyrrole containing systems offer possibilities as novel metalbinding macrocycles.²,4,5,13b,35,14 For instance, suitably designedsystems could act as versatile ligands capable of binding larger metalcations and/or stabilizing higher coordination geometries² than thoseroutinely accommodated within the normally tetradentate ca. 2.0 Å radiusporphyrin core.²¹ The resulting complexes could have importantapplication in the area of heavy metal chelation therapy, serve ascontrast agents for magnetic resonance imaging (MRI) applications, actas vehicles for radioimmunological labeling work, or serve as newsystems for extending the range and scope of coordinationchemistry.¹⁴,39 In addition, the free-base (metal-free) and/ordiamagnetic metal-containing materials could serve as usefulphotosensitizers for photodynamic therapeutic applications. In recentyears a number of pentadentate polypyrrolic aromatic systems, includingthe "sapphyrins",³,6 "oxosapphyrins",⁷ "smaragdyrins",³,6 "platyrins",⁸and "pentaphyrin"⁹ have been prepared and studied as their metal-freeforms. For the most part, however, little or no information is availablefor the corresponding metallated forms. Prior to this invention theuranyl complex of "superphthalocyanine" was the only metal-containingpentapyrrolic system which has been prepared and characterizedstructurally.² The "superphthalocyanine" system is not capable ofexistence in either its free-base or other metal-containing forms.²Thus, prior to the present invention, no versatile, structurallycharacterized, pentadentate aromatic ligands were available,^(13b)although a number of nonaromatic pyridine-derived pentadentate systemshad previously been reported.³⁷,38

Gadolinium(III) complexes derived from strongly binding anionic ligands,such as diethylenetriamine pentaacetic acid (DTPA),⁴⁰⁻⁴²1,4,7,10-tetraazacyclododecane N,N',N", N'"-tetraacetic acid(DOTA),⁴⁰,43,44 and1,10-diaza-4,7,13,16-tetraoxacyclooctadecane-N,N'-diacetic acid(dacda),⁴⁰,45 are among the most promising of the paramagnetic contrastagents currently being developed for use in magnetic resonance imaging(MRI)⁴⁰ The complex, [GdDTPA]⁻, is now being used clinically in theUnited States in certain enhanced tumor detection and other imagingprotocols.⁴⁰ Nonetheless, the synthesis of other gadolinium(III)complexes remains of interest since such systems might have greaterkinetic stability, superior relaxivity, or better biodistributionproperties than this or other carboxylate-based contrast agents. Thewater-soluble porphyrin derivatives, such as tetrakis(4-sulfonatophenyl) porphyrin (TPPS) cannot accommodate completely thelarge gadolinium(III) cation⁴⁷ within the relatively small porphyrinbinding core (r≅2.0 Å⁴⁸), and, as a consequence, gadolinium porphyrincomplexes are invariably hydrolyrically unstable.³³,34,46,49,50. Largerporphyrin-like ligands may offer a means of circumventing thisproblem.⁵¹⁻⁵⁹

A promising new modality for use in the control and treatment of tumorsis photodynamic therapy (PDT).⁶⁰⁻⁶⁴ This technique uses of aphotosensitizing dye, which localizes at, or near, the tumor site, andwhen irradiated in the presence of oxygen serves to produce cytotoxicmaterials, such as singlet oxygen (O₂ (¹ Δ_(g))), from benign precursors(e.g. (O₂ (³ Σ_(g) -)). Diamagnetic porphyrins and their derivatives arethe dyes of choice for PDT. It has been known for decades thatporphyrins, such as hematoporphyrin, localize selectively in rapidlygrowing tissues including sarcomas and carcinomas.⁶⁵ The hematoporphyrinderivative (HPD),⁶¹⁻⁶⁴,66-80 is an incompletely characterized mixture ofmonomeric and oligomeric porphyrins.⁸¹⁻⁸⁶ The oligomeric species, whichare believed to have the best tumor-localizing ability,⁸²,85 aremarketed under the trade name Photofrin II® (PII) and are currentlyundergoing phase III clinical trials for obstructed endobronchial tumorsand superficial bladder tumors. The mechanism of action is thought to bethe photoproduction of singlet oxygen (O₂ (¹ Δ_(g))), althoughinvolvement of superoxide anion or hydroxyl and/or porphyrin-basedradicals cannot be entirely ruled out.⁸⁷⁻⁹² Promising as HPD is, it andother available photosensitizers (e.g., the phthalocyanines andnaphthalocyanines) suffer from serious disadvantages.

While porphyrin derivatives have high triplet yields and long tripletlifetimes (and consequently transfer excitation energy efficiently totriplet oxygen),^(101b),g their absorption in the Q-band regionparallels that of heme-containing tissues. Phthalocyanines andnaphthalocyanines absorb in a more convenient spectral range but havesignificantly lower triplet yields;¹⁰² moreover, they tend to be quiteinsoluble in polar protic solvents, and are difficult to functionalize.Thus the development of more effective photochemotherapeutic agentsrequires the synthesis of compounds which absorb in the spectral regionwhere living tissues are relatively transparent (i.e., 700-1000nm),^(99d) have high triplet quantum yields, and are minimally toxic.The present inventors have recently reported¹⁰³ (see Example 1) thesynthesis of a new class of aromatic porphyrin-like macrocycles, thetripyrroledimethine-derived "texaphyrins", which absorb strongly in thetissue-transparent 730-770 nm range. The photophysical properties ofmetallotexaphyrins parallel those of the corresponding metalloporphyrinsand the diamagnetic complexes sensitize the production of ¹ O₂ in highquantum yield

Acquired immunodeficiency syndrome (AIDS) is among the most seriouspublic health problems facing our nation today. AIDS, first reported in1981 as occurring among male homosexuals,⁶⁰ is a fatal human diseasewhich has now reached pandemic proportions. At present, sexual relationsand needle-sharing are the dominant mechanisms for the spread of AIDS.⁶⁰Since the testing of blood supplies began, the percentage of AIDSinfections due to blood transfusions has dropped considerably..sup.60,104-107 However, an absolutely fail-proof means must be developed toinsure that all stored blood samples are free of the AIDS virus (andideally all other blood-borne pathogens). Serologic tests for HIV-1 areinsufficient to detect all infected blood samples, in particular, thosederived from donors who have contracted the disease but not yet produceddetectable antibodies.¹⁰⁴⁻¹⁰⁷

Any blood purification procedure used to remove AIDS virus or otherblood-borne pathogens should operate without introducing undesirabletoxins, damaging normal blood components, or inducing the formation ofharmful metabolites. This precludes the use of common antiviral systemssuch as those based on heating, UV irradiation, or purely chemicalmeans. A promising approach is the photodynamic one alluded to above.Here, preliminary studies, carried out by researchers at the BaylorResearch Foundation, Dr. Matthews and his team,⁹³⁻⁹⁶ and others,⁹⁷,98have served to show that HPD and PII, in far lower dosages than arerequired for tumor treatment, act as efficient photosensitizers for thephoto-deactivation of cell-free HIV-1, herpes simplex (HSV), hepatitisand other enveloped viruses. The success of this procedure derives fromthe fact that these dyes localize selectively at or near themorphologically characteristic, and physiologically essential, viralmembrane ("envelope") and catalyze the formation of singlet oxygen uponphotoirradiation. The singlet oxygen destroys the essential membraneenvelope. This kills the virus and eliminates infectivity. Photodynamicblood purification procedures, therefore, rely on the use ofphotosensitizers which localize selectively at viral membranes, just asmore classic tumor treatments require dyes that are absorbed or retainedpreferentially at tumor sites. Simple enveloped DNA viruses like HSV-1are good models for testing putative photosensitizers for potential usein killing the far more hazardous HIV-1 retrovirus. This correspondenceholds only as far as freely circulating (as opposed to intracellular)viruses are concerned. Complete prophylactic removal of HIV-1 from bloodproducts will require the destructive removal of the virus from withinmonocytes and T lymphocytes.¹⁰⁸

This "first generation" of dyes suffers from a number of seriousdeficiencies which may militate against their eventual use in biomedicalapplications. Each of these deficiencies has important clinicalconsequences. Since HPD and PII do not contain a single chemicallywell-defined constituent, coupled with the fact that the activecomponents have yet to be identified with certainty,⁸²⁻⁸⁶ means that theeffective concentrations vary from preparation to preparation. Thus thedosage, and the light fluence, cannot be optimized and predetermined forany particular application. Since they are not metabolized rapidly,significant quantities of these dyes remain in stored blood units afterprophylactic photoinduced HIV-1 removal and remain in patients bodieslong after photodynamic tumor treatment. The latter retention problem,in particular, is known to be serious; HPD and PII localize in the skinand induce photosensitivity in patients for weeks afteradministration..sup. 64,109 Since the longest wavelength absorptionmaximum for these dyes falls at 630 nm, most of the incipient energyused in phototreatment is dispersed or attenuated before reaching thecenter of a deep-seated tumor and as a result, little of the initiallight is available for singlet oxygen production and therapy.¹¹⁰⁻¹¹² Astudy using a mouse model with a 3 mm tumor implanted beneath the skinindicated that as much as 90% of the energy is lost by the base of thetumor.¹¹⁰ More effective treatment of deep-seated or large tumors may bepossible if photosensitizers could be developed which absorb in the >700nm region, provided, of course, they retain the desirable features ofHPD and PII (e.g. selective localization in target tissues and low darktoxicity). One aspect of the present invention involves development ofsuch improved photosensitizers for use in photodynamic tumor treatmentand blood purification protocols.

The following list summarizes features which would be desirable inbiomedical photosensitizers:

1. Easily available

2. Low intrinsic toxicity

3. Long wavelength absorption

4. Efficient photosensitizer for singlet oxygen production

5 Fair solubility in water

6. Selective up-take in tumor tissue and/or

7. Showing high affinity for enveloped viruses

8. Quick degradation and/or elimination after use

9. Chemically pure and stable

10. Easily subject to synthetic modification

In recent years, considerable effort has been devoted to the synthesisand study of new photosensitizers which might meet these desiderata.Although a few of these have consisted of classic dyes such as those ofthe rhodemine and cyanine classes,¹¹³⁻¹¹⁵ many have been porphyrinderivatives with extended π networks.¹¹⁶⁻¹²⁶ Included in this lattercategory are the purpurins and verdins¹¹⁶ of Morgan and otherchlorophyll-like species,¹¹⁷⁻¹¹⁹ the benz-fused porphyrins of Dolphin etel.,¹²⁰ and the sulfonated phthalocyanines and napthophthalocyaninesstudied by Ben-Hur,¹²¹ Rodgers,¹²² and others.¹²³⁻¹²⁷ Of these, only thenapthophthalocyanines absorb efficiently in the most desirable >700 nmspectral region. These particular dyes are difficult to prepare in achemically pure, water soluble form and are relatively inefficientphotosensitizers for singlet oxygen production, perhaps even actingphotodynamically via other oxygen derived toxins (e.g. superoxide). Thusa search continues for yet a "third generation" of photosensitizerswhich might better meet the ten critical criteria listed above.

It is an important aspect of the present invention that an improved"third generation" of photosensitizers is obtained using large,pyrrole-containing "expanded porphyrins". These systems, beingcompletely synthetic, can be tuned so as to incorporate any desiredproperties. In marked contrast to the literature of the porphyrins, andrelated tetrapyrrolic systems (e.g. phthalocyanines, chlorins, etc.),there are only a few reports of larger pyrrole-containing systems, andonly a few of these meet the criterion of aromaticity deemed essentialfor long-wavelength absorption and singlet oxygen photosensitization.¹²⁸In addition to the present inventors' studies of texaphyrin 1_(B),¹²⁹(see FIGS. 1B and 2B), and "sapphyrin", first produced by the groups ofWoodward³ and Johnson⁶, there appear to be only three largeporphyrin-like systems which might have utility as photosensitizersThese are the "platyrins" of LeGoff⁸, the stretched porphycenes ofVogel^(131a) and the vinylogous porphyrins of Franck.¹³⁰ The presentstudies indicate that an expanded porphyrin approach to photodynamictherapy is promising. The porphycenes,^(131b), 131c a novel class of"contracted porphyrins" also show promise as potentialphotosensitizers.¹³²

The present invention involves a major breakthrough in the area ofligand design and synthesis. It involves the synthesis of the firstrationally designed aromatic pentadentate macrocyclic ligand, thetripyrroledimethine-derived "expanded porphyrin" 1_(B).¹²⁹ Thiscompound, to which the trivial name "texaphyrin" has been assigned, iscapable of existing in both its free-base form and of supporting theformation of hydrolyrically stable 1:1 complexes with a variety of metalcations, such as Cd²⁺, Hg²⁺, In³⁺, Y³⁺, Nd³⁺, Eu³⁺, Sm³⁺, La³⁺, Lu³⁺,Gd³⁺, and other cations of the lanthanide series that are too large tobe accommodated in a stable fashion within the 20% smaller tetradentatebinding core of the well-studied porphyrins. In addition, since thefree-base form of 1_(B) is a monoanionic ligand, the texaphyrincomplexes formed from divalent and trivalent metal cations remainpositively charged at neutral pH. As a result, many of these complexesare more water soluble than the analogous porphyrin complexes.

To date, two X-ray crystal structures of two different Cd²⁺ adducts havebeen obtained, one of the coordinatively saturated, pentagonalbipyramidal bispyridine complex;^(129a) the other of a coordinativelyunsaturated pentagonal pyramidal benzimidazole complex.^(129b) Bothconfirm the planar pentadentate structure of this new ligand system andsupport the assignment of this prototypical "expanded porphyrin" asaromatic.

Further support for the aromatic formulation comes from the opticalproperties of 1_(B) and 1_(C). The lowest energy Q-type band of thestructurally characterized bispyridine cadmium(II) adduct of complexI_(C) at 767 nm (ε=51,900) in CHCl₃ is 10-fold more intense and redshifted by almost 200 nm as compared to that of a typical referencecadmium(II) porphyrin. Compound 1_(B) and both its zinc(II) andcadmium(II) complexes are very effective photosensitizers for singletoxygen, giving quantum yields for ¹ O₂ formation of between 60 and 70%when irradiated at 354 nm in air-saturated methanol.^(129c) Relatedcongeneric texaphyrin systems bearing substituents on the tripyrroleand/or phenyl portions and incorporating La(III) and/or Lu(III) metalcenters, have been found to produce ¹ O₂ in quantum yields exceeding 70%when irradiated under similar conditions. Thus, it is this remarkablecombination of light absorbing and ¹ O₂ photosensitizing propertieswhich make these systems ideal candidates for use in photodynamictherapy and blood purification protocols.

SUMMARY OF THE INVENTION

The texaphyrin derivatives described in this continuation-in-partapplication are extensions of the structure 29_(D) in FIG. 27 of theparent application Ser. No, 771,393. By texaphyrin, we mean a compoundwith the central ring system depicted in structure 1A.

The present invention involves hydroxyl derivatives of texaphyrin, anovel tripyrrole dimethine-derived "expanded porphyrin", the synthesisof such compounds and their uses. The desirable properties ofhydroxylated derivatives of texaphyrin are:

1) appreciable solubility, particularly in aqueous media;

2) biolocalization in desired target tissue;

3) the ability to attach to solid matrices;

4) the ability to be attached to biomolecules;

5) efficient chelation of divalent and trivalent metal cations;

6) absorption of light in the physiologically important region of690-880 nm;

7) high chemical stability;

8) ability to stabilize diamagnetic complexes that form long-livedtriplet states in high yield and that act as efficient photosensitizersfor the formation of singlet oxygen.

The reduced sp³ form of the texaphyrin molecule has the structure 1_(A)shown in FIG. 1A. Upon oxidation, an aromatic structure 1_(B) is formedand upon incorporation of a metal salt, such as CdCl₂, the chelate 1_(C)or its analogue incorporating other di- or trivalent cations, is formed.The synthetic scheme for the basic texaphyrin molecule is described inFIGS. 2A and 2B. These molecules are the subject of previous patentapplications Ser. Nos. 771,393 and 539,975. The derivatives disclosed inthis invention have substituents on the benzene ring portion of themolecule referred to as B or the tripyrrole portion of the moleculereferred to as T. The number following the B or T indicates the numberof hydroxyl groups that have been incorporated into that portion of themolecule.

The present invention relates to water soluble compounds retaininglipophilicity and having the structure: ##STR1## wherein M is H, adivalent or a trivalent metal cation; wherein N is an integer between-20 and +2; and

wherein the substituents R₁, R₂, R₃, R₄, and R₅ are independently

hydrogen, [H];

hydroxyl, [OH];

alkyl groups attached via a carbon or oxygen;

hydroxyalkyl groups attached via a carbon or oxygen; these may be C_(n)H.sub.(2n+1) O_(y) or OC_(n) H.sub.(2n+1) O_(y) ; where at least one ofthe subtituents R₁, R₂, R₃, R₄, and R₅ has at least one hydroxysubstituent; where the molecular weight of any one of R₁, R₂, R₃, R₄, orR₅ is less than or equal to about 1000 daltons; where n is a positiveinteger or zero; and

where y is zero or a positive integer less than or equal to (2n+1);

oxyhydroxyalkyl groups (containing independently hydroxy substituents orether branches) attached via a carbon or oxygen; these may beC.sub.(n-x) H.sub.[(2n+1)-2x] O_(x) O_(y) or OC.sub.(n-x)H.sub.[(2n+1)-2x] O_(x) O_(y) ; where n is a positive integer or zero, xis zero or a positive integer less than or equal to n, and y is zero ora positive integer less than or equal to [(2n+1)-2x];

oxyhydroxyalkyl groups (containing independently substituents on thehydroxyls of the oxyhydroxyalkyl compounds described above or carboxylderivatives) attached via a carbon or oxygen; these may be C_(n)H.sub.[(2n+1)-q] O_(y) R^(a) _(q), OC_(n) H.sub.[(2n+1)-q] O_(y) R^(a)_(q) or (CH₂)_(n) CO₂ R^(a) ; where n is a positive integer or zero, yis zero or a positive integer less than [(2n+1)-q], q is zero or apositive integer less than or equal to 2n+1, R^(a) is independently H,alkyl, hydroxyalkyl, saccharide, C.sub.(m-w) H.sub.[(2m+1)-2w] O_(w)O_(z), O₂ CC.sub.(m-w) H.sub.[(2m+1)-2w] O_(w) O_(z) orN(R)OCC.sub.(m-w) H.sub.[(2m+1)-2w] O_(w) O_(z) ; where m is a positiveinteger or zero,w is zero or a positive integer less than or equal to m,z is zero or a positive integer less than or equal to [(2m+1)-2w], R isH, alkyl, hydroxyalkyl, or C_(m) H.sub.[(2m+1)-r] O_(z) R^(b) _(r) ;where m is a positive integer or zero, z is zero or a positive integerless than [(2m+1)-r], r is zero or a positive integer less than or equalto 2m+1, and R^(b) is independently H, alkyl, hydroxyalkyl, orsaccharide;

carboxyamidealkyl groups (containing independently hydroxyl groups, orsecondary or tertiary amide linkages) attached via a carbon or oxygen;these may be (CH₂)_(n) CONHR^(a), O(CH₂)_(n) CONHR^(a) (CH₂)_(n)CON(R^(a))₂, or O(CH₂)_(n) CON(R^(a))₂ ; where n is a positive integeror zero, R^(a) is independently H, alkyl, hydroxyalkyl, saccharide,C.sub.(m-w) H.sub.[(2m+1)-2w] O_(w) O_(z), O₂ CC.sub.(m-w)H.sub.[(2m+1)-2w] O_(w) O_(z) or N(R)OCC.sub.(m-w) H.sub.[(2m+1)-2w]O_(w) O_(z) ; where m is a positive integer or zero, w is zero or apositive integer less than or equal to m, z is zero or a positiveinteger less than or equal to [(2m+1)-2w], R is H, alkyl, hydroxyalkyl,or C_(m) H.sub.[(2m+1)-r] O_(z) R^(b) _(r) ; where m is apositiveinteger or zero, z is zero or a positive integer less than[(2m+1)-r], r is zero or a positive integer less than or equal to 2m+1,and R^(b) is independently H, alkyl, hydroxyalkyl, or saccharide; or

carboxyalkyl groups (containing independently hydroxyl groups, carboxylsubstituted ethers, amide substituted ethers or tertiary amides removedfrom the ether) attached via a carbon or oxygen; these may be C_(n)H.sub.[(2n+1)-q] O_(y) R^(c) _(q) or OC_(n) H.sub.[(2n+1)-q] O_(y) R^(c)_(q) ; where n is a positive integer or zero, y is zero or a positiveinteger less than [(2n+1)-q], q is zero or a positive integer less thanor equal to 2n+1, R^(c) is (CH₂)_(n) CO₂ R^(d), (CH₂)_(n) CONHR^(d) or(CH₂)_(n) CON(R^(d))₂ ; where n is a positive integer or zero, R^(d) isindependently H, alkyl, hydroxyalkyl, saccharide, C_(m-w))H.sub.[(2m+1)-2 w] O_(w) O_(z), O₂ CC.sub.(m-w) H.sub.[(2m+1)-2w] O_(w)O_(z) or N(R)OCC_(m-w)) H.sub.[(2m+1)-2w] O_(w) O_(z) ; where m is apositive integer or zero, w is zero or a positive integer less than orequal to m, z is zero or a positive integer less than or equal to[(2m+1)-2w], R is H, alkyl, hydroxyalkyl, or C_(m) H.sub.[(2m+1)-r]O_(z) R^(b) _(r) ; where m is a positive integer or zero, z is zero orapositive integer less than [(2m+1)-r], r is zero or a positive integerless than or equal to 2m+1, and R^(b) is independently H, alkyl,hydroxyaikyl, or saccharide;

where at least one of R₁, R₂, R₃, R₄ and R₅ has at least one hydroxysubstituent and the molecular weight of any of R₁, R₂, R₃, R₄ or R₅ isless than or equal to about 1000 daltons.

In the above-described metallic complexes M may be a divalent metal ionselected from the group consisting of Ca⁺², Mn⁺², Co⁺², Ni⁺², Zn⁺²,Cd⁺², Hg⁺², Sm⁺² and UO₂ ⁺², (and N is 1). In certain aspects M ispreferably Cd⁺² or Zn⁺² or Hg⁺². When M is a trivalent metal ion, it ispreferably selected from the group consisting of Mn⁺³, Co⁺³, Ni⁺³, Y⁺³,In⁺³, Pr⁺³, Nd⁺³, Sm⁺³, Fe⁺³, Ho⁺³, Ce⁺³, Eu⁺³, Gd⁺³, Tb⁺³, Dy⁺³, Er⁺³,Tm⁺³, Yb⁺³, Lu⁺³, La⁺³ and U⁺³ ; (and N is 2). Most preferred trivalentmetal ions are In⁺³ La⁺³, Lu⁺³, and Gd⁺³.

A preferred water solubie compound retaining lipophilicity has hydroxylgroups only in the B portion of the molecule and has the structure:##STR2## wherein M is H, a divalent or a trivalent metal cation; N is aninteger between -2 0 and +2; R₁, R₂, R₃, and R₄ are independently C_(n)H_(2n+1) where n is a positive integer; and R₅ is hydroxyl,hydroxyalkyl, oxyhydroxyalkyl, carboxyalkyl or carboxyamidealkyl; whereR₅ has at least one hydroxy substituent, and the molecular weight of anyone of R₁, R₂, R₃, R₄, or R₅ is less than or equal to about 1000daltons.

Another preferred water soluble compound retaining lipophilicity hashydroxyl groups only in the T portion of the molecule and has thestructure: ##STR3## wherein M is H, a divalent or a trivalent metalcation; N is an integer between -20 and +2; R₁, R₂, R₃, and R₄ areindependently hydroxyl, alkyl, hydroxyalkyl, oxyhydroxyalkyl,carboxyalkyl or carboxyamidealkyl; and R₅ is H or C_(n) H_(2n+1) ; whereat least one of R₁, R₂, R₃, and R₄ has at least one hydroxy substituent,the molecular weight of any one of R₁, R₂, R₃, R₄, or R₅ is less than orequal to about 1000 daltons, and n is a positive integer.

Another preferred water soluble compound retaining lipophilicity hashydroxyl groups in both the B and T portions of the molecule and has thestructure: ##STR4## wherein M is H, a divalent or a trivalent metalcation; N is an integer between -20 and +2; R₁, R₂, R₃, R₄, and R₅ areindependently H, OH, C_(n) H.sub.(2n+1) O_(y) or OC_(n) H.sub.(2n+1)O_(y) ; where at least one of R₁, R₂, R₃, and R₄ has at least onehydroxy substituent, R₅ has at least one hydroxy substituent, themolecular weight of any one of R₁, R₂, R₃, R₄, or R₅ is less than orequal to about 1000 daltons, n is a positive integer or zero, and y iszero or a positive integer less than or equal to (2n+1).

In the above described metallic complexes M may be a divalent metalliccation selected from the group consisting of Ca⁺², Mn⁺², Co⁺², Ni⁺²,Zn⁺², Cd⁺², Hg⁺², Sm⁺² and UO₂ ⁺², and N is 1. When M is a trivalentmetal cation, it is preferably selected from the group consisting ofMn⁺³, Co⁺³, Ni⁺³, Y⁺³, In⁺³, Pr⁺³, Nd⁺³, Sm⁺³, Eu⁺³, Gd⁺³, Tb⁺³, Dy⁺³,Er⁺³, Fe⁺³, Ho⁺³, Ce⁺³, Tm⁺³, Yb⁺³, Lu⁺³, La⁺³ and U⁺³ ; and N is 2.Most preferred trivalent metal ions are In⁺³, Gd⁺³, La⁺³, or Lu⁺³ and Nis +2.

A preferred water soluble compound retaining lipophilicity of thisinvention has been prepared as one having the structure with the trivialname B2 (See FIG. 6): ##STR5## wherein M is H, a divalent or trivalentmetal cation, and N is 0, 1 or 2. Particularly preferred metal cationsare Gd⁺³, Lu⁺³, La⁺³, In⁺³, and N is 2.

Another preferred water soluble compound retaining lipophilicity has thestructure with the trivial name T2: ##STR6## wherein M is H, a divalentor trivalent metal cation, and N is 0, 1 or 2. Particularly preferredmetal cations are Gd⁺³, Lu⁺³, La⁺³, or In⁺³, and N is 2.

Another preferred water soluble compound retaining lipophilicity has thestructure with the trivial name B2T2 or T2B2: ##STR7## wherein M is H, adivalent or trivalent metal cation, and N is 0, 1 or 2. Particularlypreferred metal cations are Gd⁺³, Lu⁺³, La⁺³, or In⁺³, and N is 2.

Another preferred water soluble compound retaining ipophilicity has thestructure with the trivial name B4: ##STR8## wherein M is H, a divalentor trivalent metal cation, and N is 0, 1 or 2. Particularly preferredmetal cations are Gd⁺³, Lu⁺³, La⁺³, In⁺³, and N is 2.

Another preferred water soluble compound retaining lipophilicity has thestructure with the trivial name B4T2 or T2B4: ##STR9## wherein M is H, adivalent or trivalent metal cation, and N is 0, 1 or 2. Particularlypreferred metal cations are Gd⁺³, Lu⁺³, La⁺³, In⁺³, and N is 2.

Another preferred water soluble compound retaining lipophilicity has thestructure with the trivial name B4T3 or T3B4: ##STR10## wherein M is H,a divalent or trivalent metal cation, and N is 0, 1 or 2. Particularlypreferred metal cations are Gd⁺³, Lu⁺³, La⁺³, In⁺³, and N is 2.

In the above described preferred compounds M may be a divalent metalliccation selected from the group consisting of Ca⁺², Mn⁺², Co⁺², Ni⁺²,Zn⁺², Cd⁺², Hg⁺², Sm⁺² and UO₂ ⁺², and N is 1. When M is a trivalentmetal cation, it is preferably selected from the group consisting ofMn⁺³, Co⁺³, Ni⁺³, Y⁺³, In⁺³, Pr⁺³, Nd⁺³, Sm⁺³, Eu⁺³, Gd⁺³, Tb⁺³, Dy⁺³,Er⁺³, Fe⁺³, Ho⁺³, Ce⁺³, Tm⁺³, Yb⁺³, Lu⁺³, La⁺³, and U⁺³ ; and N is 2.Most preferred trivalent metal ions are In⁺³, Gd⁺³, La⁺³, or Lu⁺³ and Nis +2.

By combining various substituted intermediates, one skilled in the artcan see how a large variety of hydroxy-substituted texaphyrins could besynthesized. Water soluble means soluble in aqueous fluids to about 1 mMor better. Retaining lipophilicity means having greater affinity forlipid rich tissues or materials than surrounding nonlipid rich tissuesor materials and in the case of viruses in suspension means affinity forthe membraneous coat of the virus. Lipid rich means having a greateramount of triglyceride, cholesterol, fatty acids or the like.Hydroxyalkyl means alkyl groups having hydroxyl groups attached.Oxyalkyl means alkyl groups attached to an oxygen. Oxyhydroxyalkyl meansalkyl groups having ether or ester linkages, hydroxyl groups,substituted hydroxyl groups, carboxyl groups, substituted carboxylgroups or the like. Saccharide includes oxidized, reduced or substitutedsaccharide. Carboxyamidealkyl means alkyl groups with hydroxyl groups,secondary or tertiary amide linkages or the like. Carboxyalkyl meansalkyl groups having hydroxyl groups, carboxyl or amide substitutedethers, ester linkages, tertiary amide linkages removed from the etheror the like.

A method for the synthesis of an aromatic pentadentate expandedporphyrin analog metal complex having at least one hydroxy substituentis an aspect of the present invention. By aromatic pentadentate expandedporphyrin analog we mean texaphyrin. This method comprises synthesizinga diformyltripyrrole having structure A; condensing said tripyrrole withan orthophenylenediamine having structure B: ##STR11## where R₁, R₂, R₃,R₄, and R₅ are independently H, OH, alkyl, oxyalkyl, hydroxyalkyl,carboxyalkyl, carboxyamidealkyl or oxyhydroxyalkyl and where at leastone of R₁, R₂, R₃, R₄, and R₅ has at least one hydroxy substituent andwhere the molecular weight of any one of R₁, R₂, R₃, R₄, or R₅ is lessthan or equal to about 1000 daltons; and oxidizing the condensationproduct to form an aromatic pentadentate expanded porphyrin analog metalcomplex having at least one hydroxy substituent. A preferreddiformyltripyrrole is2,5-bis[(5-formyl-3-hydroxyalkyl-4-alkylpyrrol-2-yl)methyl]-3,4-dialkylpyrroleor2,5-bis[(5-formyl-3-hydroxypropyl-4-methylpyrrol-2-yl)methyl]-3,4-diethylpyrrole,(7_(H), FIG. 7B); or 2,5-bis((3-ethyl-5-formyl-4-methylpyrrol-2-yl)methyl)-3,4-diethylpyrrole (6_(E),FIG. 6).

A preferred "B" portion of these molecules is synthesized fromphenylenediamine or 1,2-diamino-4,5-bis(oxyhydroxyalkyl)benzene or1,2-diamino-4,5-bis((3'-hydroxypropyl)oxy)benzene, (6_(D), FIG. 6), or1,2-diamino-4,5-bis((2,3-dihydroxypropyl)oxy)benzene, (8_(D), FIG. 8).

Said condensation product is mixed in an organic solvent with atrivalent metal salt, a Bronsted base and an oxidant; and stirred atambient temperature or heated at reflux for at least 2-24 hours to forman aromatic pentadentate expanded porphyrin analog metal complex havingat least one hydroxy substituent. A preferred Bronsted base istriethylamine; preferred oxidants are air, oxygen, platinum oxide, and2,3-dichloro-5,6-dicyano-1,4-benzoquinone and preferred organic solventsare methanol and chloroform or methanol and benzene.

The metal complexes may be associated with, depending on the metal,anywhere from 0-6 apical ligands about the encapsulated metal center.The ligands are typically some combination of acetate, chloride,nitrate, hydroxide, water, or methanol and when bound, are not readilydissociable.

The present invention involves a method of deactivating retroviruses andenveloped viruses in an aqueous fluid. Aqueous fluid may be biologicalfluids, blood, plasma, edema tissue fluids, ex vivo fluids for injectioninto body cavities, cell culture media, supernatant solutions from cellcultures and the like. This method comprises adding a water solublehydroxy-substituted aromatic pentadentate expanded porphyrin analogmetal complex retaining lipophilicity to said aqueous fluid and exposingthe mixture to light to effect the formation of singlet oxygen.Preferred metals are diamagnetic metals and a preferred metal complex isthe Lu, La or In complex of B2T2.

A method of light-induced singlet oxygen production is an aspect of thepresent invention. The method comprises the use of a water solublehydroxy-substituted aromatic pentadentate expanded porphyrin analogmetal complex retaining lipophilicity and having intrinsicbiolocalization selectivity as a photosensitizer. Preferred metals arediamagnetic metals and a preferred metal complex is the Lu, La or Incomplex of B2T2. Intrinsic biolocalization selectivity means having aninherently greater affinity for certain tissues relative to surroundingtissues.

A method of enhancement of relaxivity comprising the administration of aparamagnetic metal ion (such as gadolinium, for example) complexed witha water soluble hydroxy-substituted aromatic pentadentate expandedporphyrin analog retaining lipophilicity is an aspect of the presentinvention. A preferred complex is the Gd complex of B2T2.

A method of treating a host harboring atheroma or benign or malignanttumor cells is an aspect of the present invention. The method comprisesthe administration to a host as a first agent, a water solublehydroxy-substituted aromatic pentadentate expanded porphyrinanalog-detectable-metal complex retaining lipophilicity, said complexexhibiting selective biolocalization in such atheroma or tumor cellsrelative to surrounding tissue; determining localization sites in thehost by reference to such detectable metal, followed by theadministration to the host as a second agent a water solublehydroxy-substituted aromatic pentadentate expanded porphyrinanalog-detectable-metal complex retaining lipophilicity and havingessentially identical biolocalization property and exhibiting theability to generate singlet oxygen upon exposure to light; andphotoirradiating the second agent in proximity to said atheroma or tumorcells. The first agent is further defined as being a paramagnetic metalcomplex, said paramagnetic metal serving as said detectable metal. Inthis case, the determination of localization sites occurs by magneticresonance imaging and the second agent is a diamagnetic metal complex.The paramagnetic metal is most preferably Gd(III) and the diamagneticmetal is most preferably La(III), Lu(III) or In(III). A variation ofthis method uses as a first agent, a gamma emitting radioisotope as thedetectable-metal complex, said gamma emitting radioisotope serving assaid detectable metal; determination of localization sites occurs bygamma body scanning and is followed by photoirradiating the second agentas described above. A preferred first agent is the Gd complex of B2T2,4,5-diethyl-10,23-dimethyl-9,24-bis(3-hydroxypropyl)-16,17-(3-hydroxypropyloxy)-13,20,25,26,27-pentaazapentacyclo[20.2.1.1³,6.1⁸,11.0¹⁴,19 ]heptacosa-1,3,5,7,9,11(27),12,14(19),15,17,20,22(25),23-tridecaene and a preferred second agent is theLu, La or In complex of B2T2. Detectable as used herein means that thelocation may be found by localization means such as magnetic resonanceimaging if the metal is paramagnetic or gamma ray detection if the metalis gamma emitting or using monochromatic X-ray photon sources. Selectivebiolocalization means having an inherently greater affinity for certaintissues relative to surrounding tissues. Essentially identicalbiolocalization property means the second agent is a texaphyrinderivative having about the same selective targeting characteristics intissue as demonstrated by the first agent.

Another aspect of this invention is a method of imaging atheroma in ahost comprising the administration to the host as an agent a watersoluble hydroxy-substituted aromatic pentadentate expanded porphyrinanalog-detectable-metal complex retaining lipophilicity, said complexexhibiting selective biolocalization in such atheroma; and imaging theatheroma in the host by reference to such detectable metal. The agent ispreferably a water soluble hydroxy-substituted aromatic pentadentateexpanded porphyrin analog-paramagnetic metal complex retaininglipophilicity, said paramagnetic metal serving as said detectable metal;amd imaging of the atheroma occurs by magnetic resonance imaging. Theparamagnetic metal is preferably Gd(III). The agent is preferably the Gdcomplex of B2T2,4,5-diethyl-10,23-dimethyl-9,24-bis(3-hydroxypropyl)-16,17-(3-hydroxypropyloxy)-13,20,25,26,27-pentaazapentacyclo[20.2.1.1³,6.1⁸,11.0¹⁴,19 ]heptacosa-1,3,5,7,9,11(27),12,14(19),15,17,20,22(25),23-tridecaene.

In these methods of use, by water soluble hydroxy-substituted aromaticpentadentate expanded porphyrin analog retaining lipophilicity we meanwater soluble texaphyrins retaining lipophilicity, however, one skilledin the art would recognize that water soluble hydroxy substitutedsapphyrin metal complexes may be used in methods for generating singletoxygen. Sapphyrins compounds are disclosed in patent applications Ser.No. 454,298 and 454,301 which are incorporated by reference herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B shows a schematic representation of the reduced (1_(A))and oxidized (1_(B)) forms of the free-base "texaphyrin" and arepresentative five coordinate cadmium complex (1_(C)) derived from this"expanded porphyrin".

FIGS. 2A and 2B schematically summarizes the synthesis of texaphyrin(2_(G) also designated 1_(B) in FIG. 1B).

FIGS. 3A and 3B shows ¹ H NMR spectrum of 1_(C).NO₃ in CDCl₃. Thesignals at 1.5 and 7.26 ppm represent residual water and solvent peaksrespectively.

FIGS. 4A and 4B shows a UV-visible spectrum of 1_(C).NO₃ 1.50×10⁻⁵ M inCHCl₃.

FIGS. 5A and 5B shows metal complexes and derivatives (5_(A) -5_(E)) ofcompounds of the parent patent application.

FIG. 6 schematically summarizes the synthesis of B2TXP, 6_(F) and[LuB2TXP]²⁺,6_(G), compounds of the present invention. compounds 6_(D)and 6_(E) are claimed as intermediates in the synthesis of B2TXP in thepresent invention.

FIGS. 7A and 7B schematically summarizes the synthesis ofB2T2TXP(7_(J)), [Gd B2T2 TXP]²⁺ (7_(K)), [Lu B2T2 TXP]²⁺ (7_(L)), and[La B2T2 TXP]²⁺ (7_(M)), compounds of the present invention. Othertrivalent metal complexes analogous to those shown can be preparedincluding that of In(III). Compound 7_(H) is claimed as an intermediatein the synthesis of B2T2TXP in the present invention.

FIG. 8 schematically summarizes the synthesis of B4T2TXP (8_(F)) and [GdB4T2 TXP]²⁺ (8_(G)), compounds of the present invention. Compound 8_(D)is claimed as an intermediate in the synthesis of B4T2TXP in the presentinvention.

FIG. 9 shows mononuclear cell killing by complexes 2_(H) (M═Zn⁺²) and1_(C) without irradiation. Cell kill was determined by [3H]-Thy uptakeafter phytohemagglutinin (PHA) stimulation.

FIG. 10 shows mononuciear cell killing by 1 μg/ml complex 1_(C) andirradiation. Cell kill was determined by [³ H]-Thy uptake after PHAstimulation.

FIGS. 11A, 11B and 11C summarizes the synthesis of polyether-linkedpolyhydroxylated texaphyrins. Ts is a tosyl group.

FIG. 12 summarizes the synthesis of catechol (i.e. benzene diol)texaphyrin derivatives bearing further hydroxyalkyl substituents off thetripyrrane-derived portion of the macrocycle.

FIG. 13 provides an example of a saccharide substituted texaphyrin inwhich the saccharide is appended via an acetal-like glycosidic linkage.Triflate is trifluoromethanesulfonate.

FIG. 14 summarizes the synthesis of a doubly carboxylated texaphyrinsystem in which the carboxyl groups are linked to the texaphyrin corevia aryl ethers or functionalized alkyl substituents. The products ofthis scheme, compounds 14_(H) and 14_(J) could be converted on tovarious esterified products wherein the ester linkages serve to appendfurther hydroxyl-containing substituents.

FIGS. 15A and 15B summarizes the synthesis of polyhydroxylatedtexaphyrin derivatives via the use of secondary amide linkages. DCC isdicyclohexylcarbodiimide, DMF is dimethylformamide, and DME isdimethoxyethane.

FIGS. 16A and 16B summarizes the synthesis of another set ofpolyhydroxyl substituted texaphyrin derivatives using similar amidebonds as in FIGS. 15A and 15B.

FIGS. 17A and 17B summarizes the synthesis of saccharide substitutedtexaphyrins, wherein the saccharide moieties are appended via amidebonds.

FIG. 18 summarizes the synthesis of polyhydroxylated texaphyrinderivatives containing branched polyhydroxyl (polyol) subunits appendedto the texaphyrin core via aryl ethers.

FIGS. 19A and 19B summarizes how similar polyol subunits may be appendedvia ester linkages.

DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention involves the synthesis and utility of novel watersoluble hydroxy-substituted aromatic pentadentate expanded porphyrinanalog metal complexes retaining lipophilicity, in particular,hydroxy-substituted texaphyrin metal complexes. The presence in thisstructure of a near circular pentadentate binding core which is roughly20% larger than that of the porphyrins, coupled with the realizationthat almost identical ionic radii pertain for hexacoordinate Cd²⁺(r=0.92 Å) and Gd³⁺ (r=0.94 Å),³⁰ prompted exploration of the generallanthanide binding properties of this monoanionic porphyrin-like ligand.The synthesis and characterization of a water-stable gadolinium (III)complex derived formally from a 16,17-dimethyl substituted analogue ofthe original "expanded porphyrin" system is described, as well as thepreparation and characterization of the corresponding europium(III) andsamarium(III) complexes.

The aromatic "texaphyrin" system described herein provides an importantcomplement to the existing rich coordination chemistry of porphyrins.For instance, by using methods similar to those described, zinc(II),manganese(II) , mercury(II), Iron(III), neodymium(III), samarium(III),gadolinium(III), lutetium(III), indium(III) , and lanthanum(III)complexes have been prepared and characterized.

The present invention involves hydroxy substituted derivatives oftexaphyrin, and the synthesis and characterization thereof. Theintroduction of hydroxy substituents on the B (benzene ring) portion ofthe molecule is accomplished by their attachment to phenylenediamine inthe 4 and 5 positions of the molecule. The introduction of hydroxysubstituents on the T (tripyrrole) portion of the molecule isaccomplished by appropriate functionalization of the alkyl substituentsin the 3 and/or 4 positions of the pyrrole rings at a synthetic stepprior to condensation with the substituted phenylenediamine. Mostpreferred derivatizations introduce substituents at the R₁ and R₂ sitesof the diformyltripyrrole (A, pg 23) and at the R₅ sites of theorthophenylenediamine (B, pg 23). Standard deprotection methodology suchas ester hydrolysis may be used to unmask the free hydroxylsubstituents. These derivatives exhibit significant solubility inaqueous media, up to 1 mM or better, yet they retain affinity for lipidrich regions which allows them to be useful in a biological environment.

The photophysical properties of the tripyrroledimethine-derived"expanded porphyrins" are reported; these compounds show strong lowenergy optical absorptions in the 690-880 nm spectral range as well as ahigh triplet quantum yield, and act as efficient photosensitizers forthe production of singlet oxygen, for example, in methanol solution.

Results indicate that these expanded porphyrin-like macrocycles areefficient photosensitizers for the destruction of free HIV-1 and for thetreatment of atheroma, benign and malignant tumors in vivo and infectedmononuclear cells in blood. Altering the polarity and electrical chargesof side groups of these macrocycles will alter markedly the degree,rate, and site(s) of binding to free enveloped viruses such as HIV-1 andto virally-infected peripheral mononuclear cells, thus modulatingphotosensitizer take-up and photosensitization of leukemia or lymphomacells contaminating bone-marrow. The use of La(III), Lu(III) or In(III)rather than Cd(II) for the production of singlet oxygen will reduce thetoxicity of these compounds in any biomedical usage. A powerfultechnique is the use of these hydroxy-substituted texaphyrins inmagnetic resonance imaging followed by photodynamic tumor therapy in thetreatment of atheroma, and benign and malignant tumors.

EXAMPLE 1

Synthesis of Compounds 1_(A) -1_(C)

This example describes the synthesis of compounds depicted in FIGS. 1A,1B, 2A and 2B; the nonaromatic methylene-bridged macrocycle 1_(A), theexpanded porphyrin named "texaphyrin" 1_(B) and the nitrate salt of thecadmium (II) complex 1_(C).

All solvents and reagents were of reagent grade quality, purchasedcommercially, and used without further purification. Sigma lipophilicSephadex (LH-20-100) and Merck type 60 (230-400 mesh) silica gel wereused for column chromatography. Melting points were recorded on aMel-temp Laboratory Devices capillary apparatus and are uncorrected.

2,5-Bis[[5-(benzylorycarbonyl)-3-ethyl-4-methylpyrrol-2-yl]methyl]-3,4-diethylpyrrole(2_(C), FIG. 2A). 3,4-Diethylpyrrole (2_(A), FIG. 2A)²⁸ (0.6 g, 4.9mmol), benzyl 5-(acetoxymethyl)-3-methyl-4-ethyl-pyrrole-2-carboxylate(2_(B), FIG. 2A)²⁹ (2.5 g, 7.9 mmol), and p-toluenesulfonic acid (0.15g) were dissolved in 60 mL of absolute ethanol and heated at 60° C. for8 h under nitrogen. The resulting suspension was reduced in volume to 30mL and placed in the freezer for several hours. The product was thencollected by filtration, washed with a small amount of cold ethanol, andrecrystallized from dichloromethane-ethanol to afford a white powder(2.07 g, 82%): mp 211° C. NMR spectra and high resolution mass spectraldata were obtained as described and are reported ^(13a).

2,5-Bis[(3-ethyl-5-formyl-4-methylpyrrol-2-yl)methyl]-3,4-diethylpyrrole(2_(E), FIG. 2A and 2B). The above diester (2_(C)) (4.5 g, 7.1 mmol) wasdissolved in 500 mL of dry THF containing 1 drop of triethylamine andhydrogenated over 5% palladium-charcoal (250 mg) at 1 atm H₂ pressureuntil the reaction was deemed complete by TLC. The catalyst wasseparated and the solution was taken to dryness on the rotaryevaporator. Recrystallization from dichloromethane-hexane yielded 2_(E)(3.2 g, quantitative) as a white powder which quickly develops a red hueupon standing in air: mp 111°-115° C. dec. The above diacid (3 g, 6.6mmol) was dissolved in 5 mL of freshly distilled trifuoroacetic acid andheated at reflux for 5 min under nitrogen and allowed to cool to roomtemperature over the course of 10 min. The above heating and coolingsequence was repeated once more and the resulting dark oil was thencooled in an ice-salt bath. Freshly distilled triethylorthoformate (5mL) was then added dropwise with efficient stirring. After 10 min thesolution was poured into 300 mL of ice water and let stand 30 min. Thedark red precipitate was collected by filtration and washed well withwater. Ethanol (ca. 50 mL) was then used to wash the precipitate fromthe filter funnel into 350 mL of 10% aqueous ammonia. The resultingyellow suspension was stirred well for an hour and then extracted withdichloromethane (5×150 mL). The dichloromethane extracts were washedwith water, dried over MgSO₄, and evaporated to dryness on the rotaryevaporator to give 2_(E) as an off-white mass. Two recrystallizationsfrom chloroform-ethanol gave crystalline product (1.91 g, 68%) with mp202°-203° C. NMR spectra and high resolution mass spectra data wereobtained as described and are reported ^(13a).

4,5,9,24-Tetraethyl-10,23-dimethyl-13,20,25,26,27-pentaazapentacyclo[20.2.1.1³,6.1⁸,11.0¹⁴,19]heptacosa-3,5,8,10,12,14-(19),15,17,20,22,24-undecaene (1_(A)). A.Acid-Catalysed Procedure. The diformyltripyrrane (2_(E), FIGS. 2A and2B) (105 mg, 0.25 mmol) and o-phenylenediamine (27 mg., 0.25 mmol) weredissolved, with heating, in a degassed mixture of 300 mL of dry benzeneand 50 mL of absolute methanol. Concentrated HCl (0.05 mL) was thenadded and the resulting gold solution heated at reflux for 24 h undernitrogen. After cooling, solid K₂ CO₃ (20 mg) was added and the solutionfiltered through MgSO₄. The solvent was then removed on the rotaryevaporator and the resulting product dissolved in 50 mL of CH₂ Cl₂ andrefiltered (to remove unreacted 2_(E)). Heptane (100 mL) was added tothe filtrate and the volume reduced to 50 mL on the rotary evaporatorwhereupon the flask was capped and placed in the freezer overnight. Theresulting white powder was then collected by filtration, washed withhexane, and dried in vacuo to yield 1_(A) (55 mg, 44%): mp 188°-190° C.

Metal Template Procedure. The diformyltripyrrane 2_(E) ando-phenylenediamine reactants were condensed together on a 0.25-mmolscale exactly as described above except that 1.0 equiv of eitherPb(SCN)₂ (80 mg) or UO₂ Cl₂ (85 mg) was added to the boiling solution atthe outset of the reaction. Following workup as outlined above, 68 mg(69%) and 60 mg (61%) of 1_(A) were obtained respectively for the Pb²⁺ -and UO₂ ²⁺ -catalyzed reactions. The products produced in this mannerproved identical with that prepared by procedure A. NMR spectra and highresolution mass spectra data were obtained as described and are reported^(13a).

4,5,9,24-Tetraethyl-10,23-dimethyl-13,20,25,26,27-pentaazapentacyclo[20.2.1.1³,6.1⁸,11.0¹⁴,19 ]heptacosa-1,3,5,7,9,11(27),12,14,16,18,20,22(25),23-tridocaene,free-base "texaphyrin" 1_(B). Macrocycle 1_(A) (50 mg, 0.1 mmol) wasstirred in methanol/chloroform (150 ml, v/v/2/1) in the presence ofN,N,N',N'-tetramethyl-1,8-diaminonaphthalene ("proton sponge") for oneday at room temperature. The reaction mixture was then poured into icewater. The organic layer was separated and washed with aqueous ammoniumchloride solution and then brine (a saturated solution of sodiumchloride in water). Following concentration on a rotary evaporator, thecrude material was purified by chromatography on SEPHADEX using firstpure chloroform and then chloroform/methanol (v/v/10/1) as eluents.After several faster red bands were discarded, a dark green band wascollected, concentrated in vacuo, and recrystallized fromchloroform/n-hexane. to give the sp² form of the ligand as a dark greenpowder in yields ranging from 3-12% with the better yields only beingobtained on rare occasions. Spectral data are reported in the parentpatent application Ser. No. 07/771,393.

The preparation of complex 1_(C).NO₃ was as follows: the reduced sp³form of the macrocyclic compound (1_(A)) (40 mg, 0.08 mmol) was stirredwith cadmium nitrate tetrahydrate (31 mg, 0.1 mmol) inchloroform/methanol (150 ml, v/v/=1/2) for 1 day. The dark greenreaction mixture was then concentrated and purified by chromatography onsilica gel as described above. The resulting crude material was thenrecrystallized from chloroform/n-hexane to give analytically pure1_(C).NO₃ in 27% yield. Under the reaction conditions both ligandoxidation and metal complexation take place spontaneously. Spectral dataare reported in the parent patent application Ser. No. 07/771,393.

The structure of compound I_(C) suggests that it can be formulated aseither an 18π-electron benzannelated [18]annulene or as an overall 22π-electron system; in either case an aromatic structure is defined. Theproton NMR spectrum of complex 1_(C).NO₃.(HNO₃) (see FIG. 3A) isconsistent with the proposed aromaticity. For the most part, complex1_(C).NO₃ shows ligand features which are qualitatively similar to thoseobserved for compound 1_(A). As would be expected in the presence of astrong diamagnetic ring current, however, the alkyl, imine, and aromaticpeaks are all shifted to lower field. Furthermore, the bridgingmethylene signals of compound 1_(A) (at δ 4.0)¹³ are replaced by a sharpsinglet, at ca. 9.2 ppm, ascribable to the bridging methine protons. Thechemical shift of this "meso" signal is similar to that observed forCd(OEP)¹⁶ (δ≈10.0),¹⁷ an appropriate 18 π-electron aromatic referencesystem, and is also similar to that observed for the free-base form ofdecamethylsapphyrin (δ ¹¹.5 -11.7),³ a 22 π-electron pyrrole-containingmacrocycle.

The optical spectrum of complex 1_(C).NO₃ (FIG. 4A) bears someresemblance to those of other aromatic pyrrole-containingmacrocycles³,6,7,18 and provides further support for the proposedaromatic structure. The dominant transition is a Soret-like band at 424nm (ε=72,700), which is considerably less intense than that seen forCd(OEP)(pyr)¹⁶ λ_(max) =421 nm, ε=288,000.¹⁸ This peak is flanked byexceptionally strong N- and Q-like bands at higher and lower energies.As would be expected for a larger π system, both the lowest energyQ-like absorption (λ_(max) =767.5 nm, ε=41,200) and emission (λ_(max)=792 nm)) bands of complex 1_(C).NO₃ are substantially red-shifted (byca. 200 nm!) as compared to those of typical cadmium porphyrins.¹⁸,19

The molecular structure of the bis-pyridine adduct, determined by X-raydiffraction analysis confirms the aromatic nature of the ligand.²⁰ Thecentral five nitrogen donor atoms of the complex are essentiallycoplanar and define a near circular cavity with a center-to-nitrogenradius of ca. 2.39 Å which is roughly 20% larger than that found inmetalloporphyrins.²¹ The cd atom lies in the plane of the central N₅binding core. The structure of the "expanded porphyrin" thus differsdramatically from that of CdTPP¹⁶,22 or CdTPP-(dioxane)₂,²³ in which thecadmium atom lies out of the porphyrin N₄ donor plane (by 0.58 and 0.32Å respectively). Moreover, in contrast to cadmium porphyrins, for whicha five-coordinate square-pyramidal geometry is preferred and to whichonly a single pyridine molecule will bind,²⁴ in the bis-pyridine adduct,the cadmium atom is seven-coordinate, being complexed by two apicalpyridine ligands. The configuration about the Cd atom is thus pentagonalbipyramidal; a rare but not unknown geometry for cadmium(II)complexes.²⁵

Under neutral conditions complex 1_(C) appears to be more stable thancadmium porphyrins: Whereas treatment of CdTPP or CdTPP(pyr) withaqueous Na₂ S leads to cation loss and precipitation of CdS, in the caseof complex 1_(C) no demetallation takes place. (Exposure to aqueousacid, however, leads to hydrolysis of the macrocycle.) Indeed, it hasnot been possible to prepare the free-base ligand 1_(B) bydemetallation. The tripyrroledimethine-derived free-base ligand 1_(B)was synthesized directly from 1_(A) by stirring in air-saturatedchloroform-methanol containingN,N,N',N'-tetramethyl-1,8-diaminonaphthalene.¹⁵ Although the yield islow (≦12%),²⁶ once formed, compound 1_(B) appears to be quite stable: Itundergoes decomposition far more slowly than compound 1_(A).¹³Presumably, this is a reflection of the aromatic stabilization presentin compound 1_(B). A further indication of the aromatic nature of thefree-base "expanded porphyrin" 1_(B) is the observation of an internalpyrrole NH signal at δ=0.90, which is shifted uDfield by over 10 ppm ascompared to the pyrrolic protons present in the reduced macrocycle1_(A).¹³ This shift parallels that seen when the sp³ -linked macrocycle,octaethylporphyrinogen (δ(NH)=6.9),²⁷ is oxidized to the correspondingporphyrin, H₂ OEP (δ (NH)=-3.74).¹⁷ This suggests that the diamagneticring current present in compound 1_(B) is similar in strength to that ofthe porphyrins.

EXAMPLE 2

Synthesis of compounds 5_(A) -5_(E).

The presence in texaphyrin of a near circular pentadentate binding corewhich is roughly 20% .larger than that of the porphyrins,^(13b) coupledwith the realization that almost identical ionic radii pertain forhexacoordinate Cd²⁺ (r=0.92 Å) and Gd³⁺ (r=0.94 Å),³⁰ promptedexploration of the general lanthanide binding properties of this newmonoanionic porphyrin-like ligand. The synthesis and characterization ofa water-stable gadolinium(III) complex (5_(C)) derived formally from a16,17-dimethyl substituted analogue (5_(B))³¹ of the original "expandedporphyrin" system is described in this example.

All solvents and reagents were of reagent grade quality, purchasedcommercially, and used without further purification. Sigma lipophilicSEPHADEX (LH-20-100) and Merck type 60 (230-400 mesh) silica gel wereused for column chromatography.

Compound 5_(C) is the metal adduct of ligand 5_(A) which was obtained inca. 90% yield by condensing 1,2-diamino-4,5-dimethylbenzene with2,5-Bis-(3-ethyl-5-formyl-4-methylpyrrol-2-ylmethyl)-3,4-diethylpyrroleunder acid catalyzed conditions identical to those used to prepare1_(A).^(13a) The sp³ form of ligand 5_(A) (42 mg, 0.08 mmol) was stirredwith gadolinium acetate tetrahydrate (122 mg, 0.3 mmol) and ProtonSponge™, N, N, N', N'-tetramethyl-1,8-diaminonaphthalene (54 mg, 0.25mmol) in chloroform/methanol (150 ml, v/v 1/2) for one day at roomtemperature. The dark green reaction mixture was concentrated underreduced pressure and chromatographed through silica gel (25 cm.×1.5 cm.)which was pretreated with chloroform/triethylamine (50 ml, v/v 25/1).Chloroform/triethylamine (25/1) and chloroform/methanol/triethylamine25/2.5/1 v/v) was used as eluents. A dark red band was first collectedfollowed by two green bands. The last green band, which showed a cleararomatic pattern by UV/VIS, was concentrated and recrystallized fromchloroform/n-hexane to give 14 mg (22%) of the Gd complex 5_(C).

Treatment of compound 5_(A) with Gd(OAc)₃, Eu(OAc)₃, and Sm(OAc)₃ underreaction and work-up conditions similar to those used to obtain 1_(C),then gave the cationic complexes 5_(C), 5_(D), and 5_(E), as theirdihydroxide adducts, in 22%, 33%, and 37% yields respectively. As judgedby the IR and microanalytical data, under the reaction and work upconditions, hydroxide anions serve to displace the acetate ligandspresumably present following the initial metal insertion procedure.

The new lanthanide complexes reported here are unique in several ways.For instance, as judged by fast atom bombardment mass spectrometric (FABMS) analysis, complexes 1_(C) -5_(E) are mononuclear 1:1 species, aconclusion that is further supported, by both high resolution FAB MSaccurate molecular weight determinations and combustion analysis. Inother words, we have found no evidence of 1:2 metal to ligand "sandwich"systems, or higher order combinations as are often found in the case ofthe better studied lanthanide porphyrins.³²

The electronic spectra represents a second remarkable feature of thesenew materials. The lanthanide complexes isolated to date display adominant Soret-like transition in the 435-455 nm region which isconsiderably less intense than that observed in the correspondingmetalloporphyrins,³³ and show a prominent low energy Q-type band in the760-800 nm region. This latter feature is diagnostic of this class of 22π-electron "expanded porphyrins"^(13b) and is both considerably moreintense and substantially red-shifted (by ca. 200 nm!) as compared tothe corresponding transitions in suitable reference lanthanideporphyrins (e.g., [GdTPPS]⁺, λ_(max) =575 nm³³).

Within the context of these general observations, it is interesting tonote that complexes derived from the somewhat more electron rich ligand5_(B) all display Q-type bands that are blue shifted by ca. 5-15 nm ascompared to those obtained from the original texaphyrin 1_(B)

A third notable property of complexes 5_(C) -5_(E) is their highsolubility in both chloroform and methanol. The fact that these threecomplexes are also moderately soluble (to roughly 10⁻ 3 Mconcentrations) in 1:1 (v.v.) methanol/water mixtures was of particularinterest. For instance, a 3.5×10⁻ 5 M solution of the gadolinium complex5_(C) in 1:1 (v.v.) methanol/water at ambient temperature shows lessthan 10% bleaching of the Soret and Q-type bands when monitoredspectroscopically over the course of 2 weeks. This suggests that thehalf-life for decomplexation and/or decomposition of this complex is≧100 days under these conditions. Under the conditions of the experimentdescribed above, no detectable shifts in the position of the Q-type bandare observed yet the Q-type transition of the free-base 5_(B) falls ca.20 nm to the blue of that of 5_(C). Thus, shifts in this direction wouldbe expected if simple demetalation were the dominant pathway leading tothe small quantity of observed spectral bleaching.

The strong hydrolyric stability of complexes 5_(C) -5_(E) is in markedcontrast to that observed for simple, water soluble gadoliniumporphyrins, such as [GdTPPS]⁺, which undergo water-induced demetalationin the course of several days when exposed to an aqueousenvironment.³³,34 It thus appears likely that gadolinium(III) complexesderived from the new texaphyrin ligand 5_(B), or its analogues, shouldprovide the basis for developing new paramagnetic contrast reagents foruse in MRI applications. In addition, the ease of preparation and stablemononuclear nature of complexes 5_(c) -5_(E) suggests that such expandedporphyrin ligands might provide the basis for extending further therelatively underdeveloped coordination chemistry of the lanthanides.

EXAMPLE 3

Synthesis of texaphyrin derivative B2.

Nomenclature. The trivial abbreviations assigned to the hydroxylatedderivatives of texaphyrin (TXP) in this and following examples refer tothe number of hydroxyl groups attached to the benzene ring portion (B)and the tripyrrole (T) portion of the molecule.

General Information. ¹ H and ¹³ C NMR spectra were obtained on a GeneralElectric QE-300 (300 MHz.) spectrometer. Electronic spectra wererecorded on a Beckman DU-7 spectrophotometer in CHCl₃. Infrared spectrawere recorded, as KBr pellets, from 4000 to 600 cm⁻¹ on a Nicolet 510PFT-IR spectrophotometer. Chemical ionization mass spectrometric analyses(CI MS) were made using a Finnigan MAT 4023. Low resolution and highresolution fast atom bombardment mass spectrometry (FAB MS) wereperformed with a Finnigan-MAT TSQ-70 and VG ZAB-2E instruments,respectively. A nitrobenzyl alcohol (NBA) matrix was utilized with CHCl₃as the co-solvent. Elemental analyses were performed by AtlanticMicrolab, Inc. Melting points were measured on a Mel-temp apparatus andare uncorrected.

Materials. All solvents and reagents were of reagent grade quality,purchased commercially, and used as received. Merck Type 60 (230-400mesh) silica gel was used for column chromatography. Thin-layerchromatography was performed on commercially prepared Whatman typesilica gel 60A plates.

1,2-bis((2-carboxy)ethoxy)-4,5-dinitrobenzene. 6_(B), FIG. 6. To a wellstirred solution of o-bis((3-hydroxypropyl)oxy)benzene²⁰⁷ (5.0 g, 22mmol) in 30 mL glacial acetic acid cooled to 15° C., 20 mL ofconcentrated nitric acid (70%) was added dropwise over a period of 15minutes. The temperature was held below 40° C. by cooling and properregulation of the rate of acid addition. After the addition, the yellowsolution was stirred at room temperature for 15 minutes. Here, thesolution was cooled again to 15° C. and 50 mL of fuming nitric acid(90%) was added dropwise over a period of 30 minutes. The orangesolution was brought to room temperature and stirred for approximately48 hours. After 48 hours, the reaction solution was checked by TLC,which displayed only one low R_(f) spot, the diacid. Therefore, theorange solution was poured onto 600 mL. of ice in a 1 liter beaker. Theprecipitated dinitro product was filtered, washed with water (1000 mL)until free from acid and dried in vacuo for 24 hours. The crude productwas recrystallized from acetone/n-hexanes to yield the diacid as fluffyyellow needles (4.20 grams, 55.2%). For the diacid: ¹ H NT(R (d₆-acetone) δ: 2.87 (t, 4H, OCH₂ CH₂ CO₂ H), 4.49 (t, 4H, OCH₂ CH₂ CO₂ H),7.71 (s, 2 H, Ar-H), 9-10 (br s, 2 H, CO₂ H). ¹³ C NMR (d₆ -acetone) δ:33.76, 66.57, 109.85, 137.14, 152.06, 171.51. EI MS, m/z (rel.intensity: 346 (100))

1,2-bis((3-hydroxypropyl)oxy)-4,5-dinitrobensene. 6_(C), FIG. 6. In adry 500 mL round bottom flask, equipped with a 125 mL pressure equalizeddropping funnel, 1,2-bis((2-carboxy)ethoxy)-4,5-dinitrobenzene (5.0 g,14.5 mmol) was dissolved in 50 mL dry THF (distilled over ketyl) andstirred at 10°-10° C. under nitrogen. To the resulting clear solution,120 mL of BH₃.THF (1M) was added dropwise over a period of 30 minutes.After the borane addition, the reaction mixture was stirred anadditional 5 minutes at 10° C. and then it was brought up to roomtemperature. The formation of the diol product was followed by TLC andthe reaction was deemed complete after approximately 2 hours. The boranesolution was quenched by careful addition of 65 mL of absolute methanol(Careful: frothing occurs!). After stirring the yellow solution for 30minutes, it was concentrated to a bright yellow solid on a rotaryevaporator. The crude solid was dissolved in 200 mL ethyl acetate andwashed with 4 M sodium acetate (2×100 mL), water (2×100 mL) and thenbrine (50 mL). The organic layer was dried over MgSO₄ and concentratedto dryness on a rotary evaporator. The crude product was recrystallizedfrom acetone/n-hexanes to afford 4.12 grams (90%) of orange needles. Forthe diol: mp 129°-130° C.; ¹ H NMR (CDCl₃) δ: 2.10 (p, 4 H, OCH₂ CH₂ CH₂OH), 3.81 (t, 4 H, OCH₂ CH₂ CH₂ OH), 4.28 (t, 4 H, OCH₂ CH₂ CH₂ OH),7.41 (s, 2 H, Ar-H). ¹³ C NMR (d₆ -acetone) δ: 32.52, 58.50, 67.81,107.88, 137.03, 152.47. EI MS, m/z (rel. intensity): 316 (100); HRMS(M⁺) 316.0914 (calcd. for C₁₂ H₁₆ N₂ O₈ : 316.0907).

1,2-Diamino-4,5-bis(3'-hydroxypropyl)oxybensene, 6_(D), FIG. 6. Thediamine was obtained by reduction of the corresponding1,2-bis((3-hydroxypropyl)oxy)-4,5-dinitrobenzene (3.0 g, 9.6 mmol) withhydrazine hydrate (4.7 mL, 96.2 mmol) and 10% palladium on carbon (200mg) in 120 mL refluxing absolute ethanol. The resulting brown suspensionbubbled for approximately 15-20 minutes and then turned colorless after1 hour. At this point, the reduction was deemed complete as judged byTLC (a low R_(f) spot). The reaction solution was hot filtered throughcelite into a dry flask, covered with aluminum foil, and thenconcentrated to a gray solid. The diamine was recrystallized from hotacetone/n-hexanes to yield 2.20 grams (91%) of an off-white fine powder.For the diamine: mp 115°-117° C.; ¹ H NMR (d₆ -DMSO) δ: 1.76 (p, 4 H,OCH₂ CH₂ CH₂ OH), 3.53 (q, 4 H, OCH₂ CH₂ CH₂ OH), 3.82 (t, 4 H, OCH₂ CH₂CH₂ OH), 4.06 (s, 4 H, NH), 4.44 (t, 2 H, OH), 6.25 (s, 2 H, ArH). ¹³ CNMR (d₆ -DMSO) δ: 42.68, 67.84, 77.08, 114.95, 139.01, 150.63. EI MS,m/z (rel. intensity): 256 (100); HRMS (M⁺) 256.1420 (calcd for C₁₂ H₂₀N₂ O₄ : 256.1423).

4,5,9,24-Tetraethyl-16,17-bis((3-hydrorypropyl)oxy)-10,23-dimethyl-13,20,25,26,27-pentaazapentacyclo[20.2.1.1³,6.1⁸,11.0¹⁴,19]-heptacosa-3,5,8,10,12,14(19), 15,17,20,22,24-undecaene. sp³ B2 TXP,6_(F), FIG. 6. This macrocycle was prepared in >90% yield from1,2-diamino-4,5-bis((3-hydroxypropyl)oxy)benzene and2,5-bis((3-ethyl-5-formyl-4-methylpyrrol-2-yl)methyl)-3,4-diethylpyrrole by using the acid-catalyzed procedure reported earlier for thepreparation of the reduced sp³ texaphyrin, see Example 1. For B2 sp³texaphyrin: mp 190° C. dec; ¹ H NMR (CDCl₃) δ: 1.05 (t, 6 H, CH₂ CH₃),1.12 (t, 6 H, CH₂ CH₃), 2.00 (t, 4 H, OCH₂ CH₂ CH₂ OH), 2.28 (s, 6 H,pyrr-CH₃), 2.35 (q, 4 H, CH₂ CH₃), 2.48 (q, 4 H CH₂ CH₃), 3.00-3.50 (bs,2 H, OH), 3.78 (t, 4 H, OCH₂ CH₂ CH₂ OH), 3.93 (s, 4 H, (pyrr)₂ --CH₂),4.19 (s, 4 H, OCH₂ CH₂ CH₂ OH), 7.16 (s, 2 H, ArH), 8.34 (s, 2 H, CHN),11.16 (s, 1 H, NH), 12.04 (s, 2 H, NH); ¹³ C NMR (CDCl₃) δ: 9.65, 15.45,16.61, 17.23, 17.60, 22.18, 31.71, 60.75, 68.58, 100.86, 120.23, 120.37,124.97, 125.06, 130.05, 133.86, 140.16, 140.86, 147.62; UV/vis λ_(max)369 nm; CI MS (M⁺) 642; CI HRMS (M⁺) 642.4039 (calcd for C₃₄ H₄₃ N₅ O₂ :642.4019).

Lutetium (III) complex of

4,5,9,24-tetraethyl-16,17-bis((3-hydroxypropyl)oxy)-10,23-dimethyl-13,20,25,26,27-pentaazapentacyclo[20.2.1.1³,6.1⁸,11.0¹⁴,19 ]heptacosa-1,3,5,7,9,11(27),12,14(19),15,17,20,22(25),23-tridecaene [LuB2Txp]²⁺ 6_(G), FIG. 6. A mixture ofthe reduced texaphyrin ligand,4,5,9,24-tetraethyl-16,17-bis((3-hydroxypropyl)oxy)-10,23-dimethyl-13,20,25,26,27-pentaazapentacyclo[20.2.1.1³,6.1⁸,11.0¹⁴,19]heptacosa 3,5,8,10,12,14(19),15,17,20,22,24-undecaene (100 mg., 0.16mmol), lutetium (III) nitrate hydrate (177 mg, 0.47 mmol) andtriethylamine (10 drops) were combined in 150 mL of refluxing methanolfor 12-24 hours. The dark green reaction mixture was concentrated on arotary evaporator to dryness and dried in vacuo for 24 hours. The crudecomplex was dissolved in a 100 mL 1:1 (v/v) mixture of chloroform andmethanol, filtered through celite and concentrated to 20 mL. A smallamount of silica gel (approx. 3 grams) was added to the flask and thenthe dark green solution was carefully concentrated to dryness on arotary evaporator. The silica was dried for 2 hours in vacuo, then itwas loaded on a chloroform packed silica column and the complex waspurified by first using neat chloroform and then increasingconcentrations of methanol in chloroform (0%-20%) as eluents. The darkgreen band collected from the column was concentrated to dryness on arotary evaporator and recrystallized from chloroform/methanol/diethylether to yield 50 mg (ca. 35%) of the lutetium (III) B2 texaphyrin. Forthe Lu (III) complex: ¹ H NMR (CDCl₃ /CD₃ OH) δ: 1.82-1.91 (m, 12 H, CH₂CH₃), 2.39 (m, 4 H, OCH₂ CH₂ CH₂ OH), 3.32 (m, 4 H, OCH₂ CH₂ CH₂ OH),3.39 (s, 6 H, pyrr-CH₃), 3.92-4.04 (m, 12 H, OCH₂ CH₂ CH₂ OH and CH₂CH₃), 9.52 (s, 2 H, CH═C), 10.24 (s, 2 H, ArH), 12.23 (s, 2 H, CH═N);UV/vis: λ_(max) 420.0, 477.5, 730.0; FAB MS M⁺ 811.

Other lanthanide and rare earth-like metal complexes may be synthesizedincluding the Gd⁺³, Lu⁺³, La⁺³, In⁺³, and Dy⁺³ complexes.

EXAMPLE 4

Synthesis of B2T2 TXP, see FIGS. 7A and 7B.

2,5-Bis[(5-bensyloxycarbonyl-4-methyl-3-methoxycarbonylethylpyrrol-2-yl)methyl]-3,4-diethylpyrrole.7_(C), FIG. 7A. In a 500 mL round bottom flask was placed 250 mL ofethanol from an unopened bottle and this was then purged with drynitrogen for ten minutes. 3,4-Diethylpyrrole 7_(B) (1.29 g, 0.01 mol)and2-acetoxymethyl-5-benzyloxycarbonyl-4-methyl-3-methoxycarbonylethylpyrrole7_(A) (7.83 g, 0.02 mol) were added and the mixture heated until all ofthe pyrroles dissolved. p-Toluenesulfonic acid (65 mg) was added and thereaction temperature maintained at 60° C. The reaction slowly changedcolor from a clear yellow to a dark red with the product precipitatingout of the solution as the reaction progressed. After ten hours thereaction was cooled to room temperature, the volume reduced to one halfon a rotary evaporator, and then placed in the freezer for severalhours. The product was collected by filtration, washed with a smallamount of cold ethanol to afford 4.61 g of an off white fine powder(61%): ¹ H NMR (CDCl₃, 250 MHz): δ 1.14 (6 H, t, CH₂ CH₃), 2.23 (6 H, s,pyrrole-CH₃), 2.31 (4 H, t, CH₂ CH₂ CO₂ CH₃), 2.50 (4 H, q, CH₂ CH₃),2.64 (4 H, t, CH₂ CH₂ CO₂ CH₃), 3.60 (10 H, br s, CH₃ CO₂ --and(pyrrole)₂ --CH₂), 4.44 (4 H, br s, C₆ H₅ CH₂), 6.99-7.02 (4 H, m,aromatic), 7.22-7.26 (6 H, m, aromatic), 8.72 (1 H, s, NH), 10.88 (2 H,br s, NH); ¹³ C NMR (CDCl₃, 250 MHz): δ 10.97, 16.78, 17.71, 19.40,22.07, 35.09, 51.46, 65.32, 117.37, 119.34, 122.14, 126.58, 126.79,127.36,.128.19, 133.55, 136.62, 162.35, 173.49; CI MS (M+H)⁺ 750; HRMS749.3676 (calc. for C₄₄ H₅₁ N₃ O₈ : 749.3676).

2,5-Bis[(5-benzyloxycarbonyl-3-hydroxypropyl-4-methylpyrrol-2-yl)methyl]-3,4-diethylpyrrole.7_(D), FIG. 7A.2,5-Bis[(5-benzyloxycarbonyl-4-methyl-3-methoxycarbonylethylpyrrol-2-yl)methyl]-3,4-diethylpyrrole7_(C) (5.00 g, 0.007 mol) was placed in a three necked 100 mL roundbottom flask and vacuum dried for at least 30 minutes. The flask wasequipped with a thermometer, an addition funnel, a nitrogen inlet tube,and a magnetic stir bar. After the tripyrrane was partially.dissolvedinto 10 mL of dry THF, 29 mL of borane (1M BH₃ in THF) was addeddropwise with stirring. The reaction became mildly exothermic and wascooled with a cool water bath. The tripyrrane slowly dissolved to form ahomogeneous orange solution which turned to a bright fluorescent orangecolor as the reaction went to completion. After stirring the reactionfor one hour at room temperature, the reaction was quenched by addingmethanol dropwise until the vigorous effervescence ceased. The solventswere removed under reduced pressure and the resulting white solidredissolved into CH_(Cl) ₂. The tripyrrane was washed three times with0.5M HCl (200 mL total), dried over anhydrous K₂ CO₃, filtered, and theCH₂ Cl₂ removed under reduced pressure until crystals of the tripyrranejust started to form. Hexanes (50 mL) was added and the tripyrraneallowed to crystallize in the freezer for several hours. The product wasfiltered and again recrystallized from CH₂ Cl₂ /ethanol. The product wascollected by filtration and vacuum dried to yield 3.69 g of an orangishwhite solid (76%): mp 172°-173° C.; ¹ H NMR (CDCl₃, 300 MHz): δ 1.11 (6H, t, CH₂ CH₃), 1.57 (4 H, p, CH₂ CH₂ CH₂ OH), 2.23 (6 H, s,pyrrole-CH₃), 2.39-2.49 (8 H, m, CH₂ CH₃ and CH₂ CH₂ CH₂ OH), 3.50 (4 H,t, CH₂ CH₂ CH₂ OH), 3.66 (4 H, s, (pyrrole)₂ --CH₂), 4.83 (4 H, s, C₆ H₅--CH₂), 7.17-7.20 (4 H, m, aromatic), 7.25-7.30 (6 H, m, aromatic), 8.64(1 H, s, NH), 9.92 (2 H, s, NH); ¹³ C NMR (CDCl₃, 300 MHZ): δ 10.97,16.72, 17.68, 20.00, 22.38, 33.22, 62.01, 65.43, 117.20, 119.75, 120.72,122.24, 127.23, 127.62, 128.30, 132.95, 136.60, 162.13; FAB MS (M⁺) 693.

2,5-Bis[(3-acetoxypropyl-5-bensyloxycarbonyl-4-methylpyrrol-2-yl)methyl]-3,4-diethylpyrrole.7_(E), FIG. 7A.2,5-Bis[(5-benzyloxycarbonyl-3-hydroxypropyl-4-methylpyrrol-2-yl)methyl]-3,4-diethylpyrrole7_(D) (36.4 g, 0.05 mol) was placed in a 1 L three necked round bottomflask and dried under vacuum for at least 30 minutes. The flask wasequipped with a dropping funnel, a thermometer, a nitrogen inlet tube,and a magnetic stir bar. CH₂ Cl₂ (600 mL dried over CaH₂) was added tothe tripyrrane and stirred under nitrogen to form an orange suspension.Pyridine (10.5 mL) was added directly to the flask followed by acetylchloride (9.5 mL) in 50 mL of dry CH₂ Cl₂ which was added dropwise fromthe addition funnel at such a rate that the temperature of the reactiondidn't exceed 25° C. An ice/water bath was used to cool the reaction.The tripyrrane slowly dissolved as the acetyl chloride was added to forma dark red homogeneous solution. The reaction was stirred at roomtemperature for approx. 3 hours then quenched with sat. aq. NaHCO₃. Theorganic layer was separated, washed three times with 0.5M HCl, then oncewith sat. NaHCO₃. The organic layer was separated, dried over MgSO₄,filtered, then reduced to dryness on the rotary evaporator. The orangesolid was dried in vacuo for several hours then redissolved into CH₂ Cl₂and crystallized using hexanes. 36.8 g of an orange colored product wasobtained (89%). A purer product can be obtained by recrystallizationfrom CH₂ Cl₂ /ethanol. For tripyrrane 7_(E) : mp 127°-129° C.; ¹ H NMR(CDCl₃, 300 MHz): δ 1.14 (6 H, t, CH₂ CH₃), 1.67 (4 H, p, CH₂ CH₂ CH₂OAc), 2.04 (6 H, s, CH₃ CO₂ CH₂), 2.22 (6 H, s, pyrrole-CH₃), 2.37 (4 H,t, CH₂ CH₂ CH₂ OAc), 2.48 (4 H, q, CH₂ CH₃), 3.57 (4 H, s, (pyrrole)₂--CH₂), 3.98 (4 H, t, CH₂ CH₂ CH₂ OAc), 4.45 (4 H, s, C₆ H₅ --CH₂),7.01-7.03 (4 H, m, aromatic), 7.23-7.29 (6 H, m, aromatic), 8.69 (2 H,s, NH), 10.95 (1 H, s, NH); ¹³ C NMR (CDCl₃, 300 MHz): δ 11.06, 16.89,17.74, 20.19, 20.93, 21.98, 29.70, 63.83, 65.31, 117.38, 118.81, 119.89,122.24, 126.42, 126.68, 127.24, 128.11, 133.53, 136.73, 162.62, 171.12;CI MS (M⁺) 777; HRMS (M+H)⁺, 778.4060 (calc. for C₄₆ H₅₆ N₃ O₈,778.4067).

2,5-Bis[(3-acetoxypropyl-5-carboxyl-4-methylpyrrol-2-yl)methyl]-3,4-diethylpyrrole. 7_(F), FIG. 7A2,5-Bis[(3-acetoxypropyl-5-benzyloxycarbonyl-4-methylpyrrol-2-yl)methyl]-3,4-diethylpyrrole7_(E) (15.0 g, 0.02 mol) was placed in a 500 mL side arm round bottomflask and dried under vacuum for at least 30 minutes. After dissolvingthe tripyrrane into 400 mL of dry THF, 10% Pd on carbon (0.75 g) and twodrops of triethylamine were added and the mixture stirred at roomtemperature under one atm. of H₂. After 15 hrs. celite was added to themixture and the catalyst was filtered off. The light orange solution wasreduced to one half volume under reduce pressure, then 100 mL of heptanewas added and the solution further reduced in volume until crystals ofthe tripyrrane diacid just started to appear. The tripyrrane was allowedto crystallize in the freezer for several hours and then filtered toyield a white color solid which developed a reddish hue on standing inair. 10.94 grams of product was obtained (96%): mp 146-148 dec; ¹ H NMR(CDCl₃, 300 MHz): δ 1.09 (6 H, t, CH₂ CH₃), 1.76 (4 H, p, CH₂ CH₂ CH₂OAc), 2.03 (6 H, s, CH₃ CO₂), 2.23 (6 H, s, pyrrole-CH₃), 2.42 (4 H, q,CH₂ CH₃), 2.49 (4 H, t, CH₂ CH₂ CH₂ OAc), 3.77 (4 H, s, (pyrrole)₂--CH₂), 4.01 (4 H, t, CH₂ CH₂ CH₂ OAc), 8.23 (1 H, s, NH), 9.29 (2 H, s,NH); FAB MS (M⁺) 597.

2,5-Bis[(3-acetoxypropyl-5-formyl-4-methylpyrrol-2-yl)methyl]-3,4-diethylpyrrole. 7_(G), FIG. 7A and 7B.2,5-Bis[(3-acetoxypropyl-5-carboxyl-4-methylpyrrol-2-yl)methyl]-3,4-diethylpyrrole7_(F) (5.80 g, 0.0097 mol) was placed in a 250 mL round bottomed flaskequipped with a nitrogen inlet and a magnetic stir bar. At roomtemperature under nitrogen trifluoroacetic acid (16 mL) was added to thetripyrrane dropwise via syringe. The tripyrrane dissolved with visibleevolution of CO₂ to form a dark orange solution. The reaction wasstirred at room temperature for 10-15 minutes, then cooled to -20° C.using a dry ice/CCl₄ bath. Freshly distilled triethylorthoformate (16mL, dried over CaH₂) was added dropwise via syringe to produce a deepred solution which was stirred an additional ten minutes at -20° C. Thecold bath was removed and 100 mL of water was added slowly to thesolution. A precipitate formed during addition of the water and theresulting orange suspension was stirred at room temperature for 20-30minutes. The product was collected by filtration, washed several timeswith water, and resuspended in 1:1 50% aqueous NH₄ OH/Ethanol (240 mL).The yellow/brown suspension was stirred for one hour at roomtemperature, filtered, washed several times with water and then washedwith a small amount of cold ethanol. The tripyrrane was recrystallizedfrom CH₂ Cl₂ /ethanol to yield 4.50 g of a reddish color solid (82%): mp179°-181° C.; ¹ H NMR (CDCl₃, 300 MHz): δ 1.11 (6 H, t, CH₂ CH₃), 1.67(4 H, p, CH₂ CH₂ CH₂ OAc), 2.05 (6 H, s, CH₃ CO₂ --), 2.19 (6 H, s,pyrrole-CH₃), 2.42-2.49 (8 H, m, CH₂ CH₃ and CH₂ CH₂ CH₂ OAc), 3.83 (4H, s, (pyrrole)₂ --CH₂), 3.99 (4 H, t, CH₂ CH₂ CH₂₀ OAc), 9.07 (2 H, s,CHO), 9.42 (1 H, s, NH), 10.70 (2 H, s, NH); ¹³ C NMR (CDCl₃, 300 MHz):δ 8.75, 16.55, 17.62, 19.98, 20.85, 22.56, 29.04, 63.71, 120.26, 121.41,121.65, 128.02, 132.81, 138.52, 171.08, 175.38; CI MS (M+1)⁺ 567; HRMS(M+H)⁺ 566.3208 (calc for C₃₈ H₄₄ N₃ O₆, 566.3230).

2,5-Bis[(5-formyl-3-hydroxypropyl-4-methylpyrrol-2-yl)methyl]-3,4-diethylpyrrole. 7_(H), FIG. 7B.2,5-Bis[(3-acetoxypropyl-5-formyl-4-methylpyrrol-2-yl)methyl]-3,4-diethylpyrrole7_(G) (5.98 g, 0.011 mol) and LiOH (1.76 g, 0.042 mol) were added to 400mL of 95% methanol, which had been degassed with nitrogen prior to use,and the mixture heated to reflux under a nitrogen atmosphere. Thereaction became homogeneous when heated. After heating for 1.25 hours,the reaction was allowed to cool to room temperature. The productprecipitated as a tan color solid as the reaction cooled. The volume ofthe reaction mixture was reduced to 75 mL on a rotary evaporator and theresulting slurry placed in the freezer for several hours. The productwas filtered and then purified by forming a slurry with 400 mL ofmethanol and 50 mL of water and heating close to boiling. The slurry wasfirst cooled to room temperature, reduced to 1/2 volume under reducedpressure, and placed in the freezer for several hours. The product wascollected by filtration and vacuum dried to yield 4.96 g of a tan powder(94%): ¹ H NMR (CD₃ OD, 300 MHz): δ 0.96 (6 H, t, CH₂ CH₃), 1.49 (4 H,p, CH₂ CH₂ CH₂ OH), 2.25 (6 H, s, pyrrole-CH₃), 2.32-2.43 (8 H, m, CH₂CH₃ and CH₂ CH₂ CH₂ OH), 3.46 (4 H, t, CH₂ CH₂ CH₂ OH), 3.85 (4 H, s,(pyrrole)₂ --CH₂),9.34 (2 H, s, CHO); CI MS (M⁺) 480; HEMS (M)⁺ 481.2942(calc for C₂₈ H₃₉ N₃ O₄, 481.2941).

4,5-Diethyl-10,23-dimethyl-9,24-bis(3-hydroxypropyl)-16,17-bis(3-hydroxypropyloxy)-13,20,25,26,27-pentaazapentacyclo[20.2.1.1³,6.1⁸,11.0.sup.14,19]heptacosa-3,5,8,10,12,14(19), 15,17,20,22,24-undecaene. 7_(J), FIG. 7B.2,5-Bis[(5-formyl-3-hydroxypropyl-4-methylpyrrol-2-yl)methyl]-3,4-diethylpyrrole7_(H) (1.00 g, 0.002 mol) and1,2-diamino-4,5-bis(3-hydroxypropyloxy)benzene 7_(X) (0.52 g, 0.002 mol)were placed in a 2 L round bottom flask with 1000 mL of toluene and 200mL of methanol. The solvents were purged with nitrogen prior to use.Concentrated HCl (0.5 mL) was added and the reaction heated to refluxunder nitrogen. The reaction went from a clear suspension of startingmaterials to a dark red homogeneous solution as the reaction proceeded.After 10 hours the reaction was cooled to room temperature and thesolvents removed under reduced pressure until the product precipitatedout of solution. The remainder of the solvent was decanted off and themacrocycle dried under vacuum. The dark red product was used withoutfurther purification (90-100%): mp 181° C.-dec; ¹ HNMR (CD₃ OD, 300MHz): δ 1.11 (6 H, t, CH₂ CH₃), 1.76 (4 H, p, pyrrole-CH₂ CH₂ CH₂ OH),2.03 (4 H, p, OCH₂ CH₂ CH₂ OH), 2.36 (6 H, s, pyrrole-CH₃), 2.46 (4 H,q, CH₂ CH₃), 2.64 (4 H, t, pyrrole-CH₂ CH₂ CH₂ OH), 3.61 (4 H, t,pyrrole-CH₂ CH₂ CH₂ OH), 3.77 (4 H, t, OCH₂ CH₂ CH₂ OH), 4.10 (4 H, s,(pyrrole)₂ --C₂), 4.22 (4 H, t, OCH₂ CH₂ CH₂ OH), 7.41 (2 H, s,aromatic), 8.30 (2 H, s, CHN); ¹³ C NMR (CD₃ OD, 300 MHz): δ 9.96,17.17, 18.65, 20.89, 24.52, 33.15, 33.45, 59.58, 61.93, 67.82, 107.11,120.66, 123.76, 124.98, 125.80, 128.68, 144.80, 144.96, 150.72, 154.60;FAB MS (M+H)⁺ 703; HRMS M⁺ 701.4120 (calc for C₄₀ H₅₅ N₅ O₆, 701.4152).

Gadolinium (III) complex of 4,5-diethyl-10,23-dimethyl-9,24-bis(3-hydrozypropyl)-16,17-(3-hydroxypropylory)-13,20,25,26,27-pentaazapentacyclo[20.2.1.1³,6.1⁸,11.0¹⁴,19]heptacosa-1,3,5,7,9,11(27),12,14(19),15,17,20,22(25),23-tridecaene.7_(K), FIG. 7B. [GdB2T2Txp]. A mixture of4,5-diethyl-10,23-dimethyl-9,24-bis(3-hydroxypropyl)-16,17-bis(3-hydroxypropyloxy)-13,20,25,26,27-pentaazapentacyclo[20.2.1.1³,6.1⁸,11.0¹⁴,19 ] heptacosa-3,5,8,10,12,14(19), 15,17,20,22,24-undecaene 7_(J) (1.52 g, 0.002 mol), gadolinium (III) acetatetetrahydrate (2.64 g, 0.007 mol), and triethylamine (ca. 1 mL) in 2 L ofmethanol was heated to reflux under air for 3.5-4 hours. The dark greenreaction was cooled to room temperature and the solvent removed underreduced pressure. Dichloromethane, containing 2% methanol, was added tothe resulting green solid to form a slurry and was filtered to wash awaysome red colored impurities (incomplete oxidation products). The complexwas then washed through the filter with methanol to leave behind someexcess gadolinium salts on the filter. The methanol was reduced to asmall volume on a rotary evaporator and then a small amount of silicagel was added. The rest of the methanol was removed carefully underreduced pressure and the complex/silica gel mixture dried under vacuumfor several hours. The silica mixture was placed on top of a silica gelcolumn and eluted with CHCl₃ containing increasing concentrations ofmethanol (5-100%). Fractions containing the complex were collected andthe solvent removed under reduced pressure. The complex was furtherpurified by passing it through a plug of neutral alumina using 1:1 CHCl₃/methanol as the eluent. The final column was used to remove anyremaining free gadolinium salts. The complex was recrystallized frommethanol/diethyl ether to yield 0.92 g of dark green powder (44%):UV/vis λ_(max),nm (CH₃ OH) 414, 474, 738, (H2O) 417, 469, 740; FAB MS(M+H)⁺ 855; HRMS, (M)⁺, 854.2995 (calc for C₄₀ H₅₀ N₅ O₆ ¹⁵⁸ Gd,854.3002).

Lanthanum (III) complex of 4,5-diethyl-10,23-dimethyl-9,24-bis(3-hydroxypropyl)-16,17-(3-hydroxypropyloxy)-13,20,25,26,27-pentaazapentacyclo]20.2.1.1³,6.1⁸,11.0¹⁴,19]heptacosa-1,3,5,7,9,11(27),12,14(19),15,17,20,22(25),23-tridecaene.7_(M), FIG. 7B. [LaB2T2Txp]. A mixture of4,5-diethyl-10,23-dimethyl-9,24-bis(3-hydroxypropyl)-16,17-bis(3-hydroxypropyloxy)-13,20,25,26,27-pentaazapentacyclo[20.2.1.1³,6.1⁸,11.0¹⁴,19 ]heptacosa-3,5,8,10,12,14(19), 15,17,20,22,24-undecaene 7_(J) (100 mg, 0.14 mmol), lanthanum (III) nitratehexahydrate (185 mg, 0.42 mmol), and triethylamine (5 drops) in methanol(150 mL) were heated to reflux under air for 16 hours. The dark greenreaction was cooled to room temperature and the solvents removed on arotary evaporator. The complex was dissolved into methanol and filteredthrough a fine glass frit. A small amount of neutral alumina was addedand the methanol removed under reduced pressure. The alumina/complexmixture was dried under vacuum for several hours then placed on top of aneutral alumina column. The column was eluted using neat CHCl₃ and CHCl₃containing increasing concentrations of methanol (5-20%). Fractionscontaining the complex were reduced to dryness on a rotary evaporatorand the resulting green solid recrystallized several times frommethanol/diethyl ether. A dark green product (66 mg) was obtained (50%):UV/vis λ_(max),nm (CH₃ OH) 417, 476, 746; FAB MS (M+H)⁺ 836; HRMS(M+H)⁺, 836.2886 (calc for C₄₀ H₅₁ N₅ O₆ ¹³⁹ La, 836.2903).

EXAMPLE 5

Synthesis of B4T2 TXP:

1,2-Dihydroxy-4,5-dinitrobenzene. 8₈, FIG. 8. In a dry 500 mL roundbottom flask, 1,2-dimethoxy-4,5-dinitrobenzene (3.2 g, 0.12 mmol) 8_(A)was stirred vigorously in 40 mL of glacial acetic acid at 30° C. Once ahomogeneous solution 200 mL of 48% HBr was added to the flask and thereaction was slowly heated to reflux. The reaction was complete asindicated by TLC after 4 hours. The work up involved pouring the cooledsolution into 800 mL of ice water and then extracting the aqueous phasewith CHCl₃ (3×150 mL) in order to remove any organic impurities. Thedinitro catechol was extracted out of the aqueous layer with ethylacetate (3×150 mL). The combined ethyl acetate extracts were washed withwater and brine (3×100 mL), then dried over MgSO₄ and concentrated to anorange residue. Approximately 100 mL of dichloromethane was added to theresidue and then placed in the freezer for several hours. The lightyellow needles that formed were filtered and washed with dichloromethaneto yield 2.37 g of product (84%). ¹ H NMR (d₆ -acetone): δ3.45 (OH),7.42 (Ar-H); ¹³ C NMR (d₆ -acetone): δ112.44, 137.00, 149.97, EI MS M⁺200.

1,2-Bis(2,3-dihydroxypropyloxy)-4,5-dinitrobensene. 8_(C), FIG. 8.1,2-Dihydroxy-4,5-dinitrobenzene 8_(B) (5.0 g, 22 mmol) and1-chloro-2,3-dihydroxypropane (12.1 g, 110 mmol) were refluxed for 48hours in a solution of potassium hydroxide (4.4 g) in 1-butanol (100 mL)under a nitrogen atmosphere. The resulting mixture was concentratedunder reduced pressure, and the dark residue was partitioned between 100mL of THF and 100 mL of brine/50 mL water solution in a 500 mLseparatory funnel. The mixture was allowed to separate and the aqueousphase was extracted with THF (2×100 mL). The combined THF extracts werewashed with brine (2×50 mL), dried over MgSO₄ and concentrated to anoily residue. Here, CH₂ Cl₂ was added very carefully to insureprecipitation of the crude product. After stirring for 15 minutes, thesuspension was filtered with a medium glass fritted funnel and air driedfor several minutes. The orange solid was taken up in 120 mL of CHCl₃and 80 mL of diethyl ether at reflux and hot filtered to remove someimpurities. The crude product was dissolved in a mixture of acetone andmethanol (sonication may be required), then 6 grams of deactivatedsilica gel was added to the orange solution. The slurry was concentratedto dryness and the orange solid was dried in vacuo for one hour. Theorange solid was loaded on a packed deactivated silica gel column. Thecolumn was eluted starting with neat CHCl₃ followed by CHCl₃ withincreasing concentration of methanol (0-10%). After a bright yellowimpurity (monoalkylated product) was removed a colorless product beganto elute (using 8-10% methanol in CHCl₃ eluents). Conversely, on TLC theproduct will elute faster than the bright yellow monoalkylated product.The purified dialkylated tetrahydroxy product can be recrystallized fromacetone/diethyl ether to yield 2.60 grams (30%) of a light yellow fluffysolid. ¹ H NMR (d₆ -acetone): δ 2.95 (bs, 4 H, OH), 3.69 (d, 4 H, OCH₂CH(OH)CH₂ OH), 4.06 (p, 2 H, OCH₂ CH(OH)CH₂ OH), 4.24-4.35 (m, 4 H, OCH₂CH(OH)CH₂ OH), 7.72 (s, 2 H, Ar-H); ¹³ C NMR (d₆ -acetone): δ 63.55,70.89, 72.53, 109.99, 137.22, 152.77. CI MS 349.

1,2-Diamino-4,5-bis((2,3-dihydroxypropyl)oxy)benzene. 8_(D), FIG. 8. Thediamine was obtained by reduction of the corresponding1,2-bis((2,3-dihydroxypropyl)oxy)-4,5-dinitrobenzene (0.30 g, 0.86 mmol)with hydrazine hydrate (1 mL) and 10% palladium on carbon (50 mg) in 40mL refluxing absolute ethanol. The resulting brown suspension bubbledfor approximately 15-20 minutes and then turned colorless after 1 hour.At this point the reduction was deemed complete as judged by TLC(Rf=0.63, 100% methanol). The reaction solution was hot filtered throughcelite into a dry flask, covered with aluminum foil, and thenconcentrated to a light yellowish oil. The diamine was taken to the nextstep without further purification. For B4 diamine: ¹ H NMR (CD₃ OD): δ3.54-3.58 (m, 4 H, OCH₂ CH(OH)CH₂ OH), 3.80-3.85 (m, 6 H, OCH₂ CH(OH)CH₂OH), 6.39 (s, 2 H, Ar-H); ¹³ C NMR (CD₃ OD): δ 64.27, 71.88, 73.22,107.61, 130.31, 143.74.

4,5-Diethyl-9,24-bis(3-hydroxypropyl)-16,17-bis((2,3-dihydroxypropyl)oxy)-10,23-dimethyl-13,20,25,26,27-pentaazapentacyclo[20.2.1.1³,6.1⁸,11.0¹⁴,19]heptacosa-3,5,8,10,12, 14(19),15,17,20,22,24-undacaene. [sp³ B4T2TXP]8_(F), FIG. 8.2,5-Bis[(5-formyl-3-hydroxypropyl-4-methylpyrrol-2-yl)methyl]-3,4-diethylpyrrole(336 mg, 0.70 mmol) and1,2-diamino-4,5-bis((2,3-dihydroxypropyl)oxy)benzene (ca 223 mg, 0.77mmol) were placed in a 1 L round bottom flask with 600 mL of toluene and175 mL of methanol. The solvents were purged with nitrogen prior to use.Concentrated HCl (ca 3 drops) was added and the reaction heated toreflux under nitrogen. After one hour the reaction was cooled to roomtemperature and the solvent removed under reduced pressure until thedark brown product precipitated. The remainder of the solvent wasdecanted off and the product dried in vacuo. The product was used in thenext step without further purification.

Gadolinium (III) complex of4,5-Diethyl-9,24-bis(3-hydroxypropyl)-16,17-bis((2,3-dihydroxypropyl)oxy)-10,23-dimethyl-13,20,25,26,27-pentaazapentacyclo[20.2.1.1³,6.1⁸,11.0¹⁴,19]heptacosa-1,3,5,7,9,11(27),12,14(19),15,17,20,22(25),23-tridecaene[GdB4T2Txp]. 8_(G), FIG. 8. Two identical reactions containing a mixtureof reduced B4T2 texaphyrin ligand,4,5-Diethyl-9,24-bis(3-hydroxypropyl)-16,17-bis((2,3-dihydroxypropyl)oxy)-10,23-dimethyl-13,20,25,26,27-pentaazapentacyclo[20.2.1.1³,6.1⁸,11.0.sup.14,19]heptacosa-3,5,8,10,12,14(19),15,17,20,22,24-undecaene, (0.75 g, 0.001mol), gadolinium (III) acetate tetrahydrate (1.19 g, 0.003 mol), andtriethylamine (ca 1 mL) were heated at reflux under air in 750 mL ofabsolute methanol. After heating for 17 hours the reactions were cooledslightly and air bubbled through the reaction mixture for severalminutes. The reactions were then heated to reflux again. After heatingfor a total of 21 hours the reactions were cooled to room temperature,the solvent removed on a rotary evaporator, and the dark green productscombined and dried in vacuo for several hours. The metal complex wasdissolved into 100 mL of methanol and 6-8 grams of deactivated silicagel was added. (The silica gel was deactivated by adding a mixture of 6mL water in 20 mL of methanol to 100 g of silica gel. After thoroughmixing, the silica gel was allowed to air dry for 12 hours beforebottling). The solvent was carefully removed on a rotary evaporator andthe silica/complex mixture dried in vacuo for one hour. The complex wasloaded onto a prepacked column of deactivated silica gel (5 cmlength×3.5 cm diameter) and eluted with chloroform containing increasingamounts of methanol (0-80%). Fractions containing the complex werecollected and concentrated to dryness. The green complex was furtherpurified by recrystallization from methanol/anhydrous ethyl ether. 480mg of product was obtained from the two combined reactions (25%). Forthe complex: UV/vis, λ_(max), nm (CH₃ OH) 415, 474, 740; FAB MS (M+H)⁺887; HR MS (M+H)⁺ 887.2977 (calc for C₄₀ H₅₁ N₅ O₈ ¹⁵⁸ Gd, 887.2981).

EXAMPLE 6

Further derivatives of Texaphyrin.

Intermediates hydroxylated in various positions can be combined toeffect the synthesis of a number of compounds. For example, the B4 TXPderivative is synthesized by reacting the intermediate compound 6_(E)from FIG. 6 with compound 8_(D) of FIG. 8. This constructs a moleculewithout hydroxyl groups on the tripyrrole moiety but with 4 hydroxylgroups on the benzene ring moiety.

The molecule T2 TXP is synthesized by reacting intermediate 7_(E) inFIG. 7 with 4,5-dimethyl-1,2-phenylenediamine to yield a texaphyrinderivative with two hydroxyls on the tripyrrole portion of the moleculeand no hydroxyl substituents on the benzene ring.

A heptahydroxylated target B4T3 TXP is obtained by using the appropriatederivative 3-hydroxypropyl-4-methylpyrrole of the pyrrole (structure7_(B) of FIG. 7) to make the trihydroxylated tripyrrole precursor whichis then reacted with compound 8_(D) of FIG. 8.

FIGS. 11A-11C, 12-14, 15A-17B, 18, 19A and 19B provide specific examplesof how one skilled in the art could extend and refine the basicsynthetic chemistry outlined in this application so as to produce otherhydroxylated texaphyrins equivalent in basic utility to thosespecifically detailed in the examples. FIGS. 11A-11C summarizes thesynthesis of polyether-linked polyhydroxylated texaphyrins. FIG. 12summarizes the synthesis of catechol (i.e. benzene diol) texaphyrinderivatives bearing further hydroxyalkyl substituents off thetripyrrane-derived portion of the macrocycle. FIG. 13 provides anexample of a saccharide substituted texaphyrin in which the saccharideis appended via an acetal-like glycosidic linkage. FIG. 14 summarizesthe synthesis of a doubly carboxylated texaphyrin system in which thecarboxyl groups are linked to the texaphyrin core via aryl ethers orfunctionalized alkyl substituents. The products of this scheme,compounds 14_(H) and 14_(J) could be converted to various esterifiedproducts wherein the ester linkages serve to append furtherhydroxyl-containing substituents. FIGS. 15A and 15B summarizes thesynthesis of polyhydroxylated texaphyrin derivatives via the use ofsecondary amide linkages. FIGS. 16A and 16B summarizes the synthesis ofanother set of polyhydroxyl substituted texaphyrin derivatives usingsimilar amide bonds as in FIGS. 15A and 15B. FIGS. 17A and 17Bsummarizes the synthesis of saccharide substituted texaphyrins, whereinthe saccharide moieties are appended via amide bonds. FIG. 18 summarizesthe synthesis of polyhydroxylated texaphyrin derivatives containingbranched polyhydroxyl (polyol) subunits appended to the texaphyrin corevia aryl ethers. FIGS. 19A and 19B summarizes how similar polyolsubunits may be appended via ester linkages.

EXAMPLE 7

Characterization of new derivatives.

New texaphyrin derivatives may be characterized fully using normalspectroscopic and analytical means, including, X-ray diffractionmethods. A complete analysis of the optical properties may be made fornew systems under a range of experimental conditions includingconditions designed to approximate those in vivo. Detailed analyses,including triplet lifetime and singlet oxygen quantum yielddeterminations may be made. The objective is to obtain a complete groundand excited state reactivity profile for each new texaphyrin produced.Questions such as when singlet oxygen production is maximized, how thequantum yield for its formation is influenced by the position of thelowest energy (Q-type) transition, whether aggregation is more prevalentin certain solvents or in the presence of certain biologically importantcomponents (e.g. lipids, proteins, etc.), and, finally, whethersignificant differences in in vitro optical properties are derived fromthe use of elaborated texaphyrins bearing cationic, anionic, or neutralsubstituents may be answered.

With newly prepared complexes, screening experiments are carried out.Standard in vitro protocols are used to evaluate the in vitrophoto-killing ability of the texaphyrin derivatives in question. Forinstance, the texaphyrin complexes of choice may be administered invarying concentrations to a variety of cancerous cells and the rate ofcell replication determined both in the presence and absence of light.Similarly, texaphyrin complexes of choice may be added to standard viralcultures and the rate of viral growth retardation determined in thepresence and absence of light. A variety of solubilizing carriers willbe used to augment the solubility and/or monomeric nature of thetexaphyrin photosensitizers and the effect, if any, that these carriershave in adjusting the biodistribution properties of the dyes will beassessed (using primarily fluorescence spectroscopy). Appropriatecontrol experiments are carried out with normal cells so that theintrinsic dark and light toxicity of the texaphyrins may be determined.

From a generalized set of in vitro experimental procedures, a clearpicture of the photodynamic capabilities of the texaphyrin derivativeswill emerge. Preliminary toxicity and stability information will resultfrom the in vitro experiments. Particular questions of interest includethe texaphyrin derivatives half life under physiological conditions,whether the nature of the central metal influences stability and whetherthe central cation is affecting cytotoxicity. As discussed in paperspublished by the present inventors,¹²⁹ it is not possible to remove thelarger bound cations (e.g. Cd²⁺ or Gd³⁺) by simple chemical means (Zn²⁺however appears to "fall out" with ease). Preliminary results indicatethat the lanthanum(III)-containing texaphyrin complex is not appreciablycytotoxic. Nonetheless, the question of intrinsic toxicity is one ofsuch central importance that the cytotoxicity of all new systems shouldbe screened in vitro and, where appropriate, further in vivo toxicitystudies carried out.

EXAMPLE 8

Viral Inactivation by Texaphyrin Macrocycles.

One aspect of the utility of the present invention is the use ofcomplexes described herein for photon-induced deactivation of virusesand virally infected or potentially infected eucaryotic cells. Thegeneral photodeactivation method used in this example was developed bythe Infectious Disease and Advanced Laser Applications Laboratories ofthe Baylor Research Foundation, Dallas, Tex. and is a subject of U.S.Pat. No. 4,878,891 which is incorporated herein by reference.

The efficiency of some of the porphyrin-like macrocycles inphotosensitized inactivation of Herpes Simplex Virus Type 1 (HSV-1) andof human lymphocytes and monocytes, both peripheral mononucleatedvascular cells (PMC) and cellular hosts of HIV-1 has been initiated.Previous studies of vital inactivation using the macrocyclicphotosensitizers dihematoporphyrin ether (DHE) or hematoporphyrinderivative (HPD) have shown that with the porphyrins, only those virusesstudied which are enveloped or possess a membraneous coat areinactivated. The enveloped viruses studied include HSV-1,cytomegalovirus, measles virus¹³³, and the human immunodeficiency virusHIV-1¹³⁴.

The photosensitized inactivation of Herpes Simplex Virus, Type 1 (HSV-1)was investigated in culture medium using various macrocycles. Resultsare listed in Table 1.

                  TABLE 1                                                         ______________________________________                                        Herpes Simplex Virus I Inactivation with                                      Expanded Porphyrin Macrocycle Complexes*                                                                % Survival Viral                                    Complex**      Conc. (μM)                                                                            Infectivity                                         ______________________________________                                        1.sub.C        20         12                                                                 10         8                                                                  2.5        20                                                                 0.25       100                                                 5.sub.B (where M = Cd)                                                                       20         4                                                                  10         14                                                                 2.5        42                                                                 0.25       100                                                 ______________________________________                                         *All light irradiation at λ max absorption and to give a light         fluence of 10 J/cm.sup.2                                                      **Structural formulas in FIGS. 1A, 1B, 5A and 5B.                        

The two cadmium-containing macrocycles (1_(C), 5_(B) (where M is Cd)),at concentrations of 20 μM demonstrated ≈90% viral inactivation asjudged by vital plaque assay.

The macrocycle photosensitizing studies employed enveloped HSV-1 as themodel for screening based on its ease of propagation and assessment ofinfectivity in cell culture. The screening procedure forphotoinactivation of HSV-1 was similar to the methods previouslydescribed.¹³⁵ Essentially, selected macrocycles at differentconcentrations were added to a cell-free suspension of 10⁶ PFU/ml ofHSV-1. The viral suspensions were irradiated at the optimal absorptionwavelength of the selected dye at different light-energy densities.Controls consisted of (1) nonirradiated virus, (2) virus irradiated inthe absence of macrocycle, and (3) virus treated with selectedconcentrations of macrocycle and maintained in the dark. All sampleswere then assessed for viral infectivity by determining the number ofPFU/ml in Vero cells.

Viral suspensions were serially diluted and subsequently absorbed ontoVero cell monolayers for 11/2 hours at 37° C. An overlay medium wasadded and the cells incubated at 37° C. for 3-4 days. The overlay mediumwas then removed, the monolayers fixed with methanol and tinctured withGiemsa, and individual plaques counted under a dissecting microscope.Uninfected cell cultures also were exposed to the macrocycle complexesto rule out direct cytotoxic effects.

The inactivation of PMC's in the absence and presence of light afterexposure to concentrations of complex 1_(C) in whole human plasmaranging from 0.015 to 38 μM is shown in FIGS. 9 and 10. Inactivation wasjudged by mitogenic assay. Toxicity onset with 1_(C) (see FIG. 1B) and2_(H) (M═Zn⁺⁺, see FIG. 2B) in the absence of light was between 0.15 and1.5 μM (FIG. 9). As shown by mitogenic assay in FIG. 10, aerobicphotosensitization of cells exposed to 1_(C) at 0.15 μM concentrationand 20 joules/cm² of 770 nm wavelength light caused significantinhibition of the cellular division of PMC's. Moderate increase ineither photosensitizer concentration or light dosage is expected toresult in essentially complete cellular inactivation.

Results indicate that the expanded porphyrin-like macrocycles should beefficient photosensitizers for free HIV-1 and infected mononuclearcells. Altering the polarity and electrical charges of side groups ofthese macrocycles is anticipated to alter the degree, rate, and perhapssite(s) of binding to free enveloped viruses such as HIV-1 and tovirally-infected peripheral mononuclear cells, thus modulatingphotosensitizer take-up and photosensitization of leukemia or lymphomacells contaminating bone-marrow as well.

EXAMPLE 9

Antibody Conjugates

Radioisotopes play a central role in the detection and treatment ofneoplastic disorders. Improving their efficacy in medical applicationsinvolves attaching radioisotopes to tumor-directed monoclonal antibodiesand their fragments. Radiolabeled antibodies could therefore serve as"magic bullets" and allow the direct transport of radioisotopes toneoplastic sites thus minimizing whole body exposure toradiation.¹⁷⁷⁻¹⁸⁷ The use of bifunctional metal chelating agents inradioimmunodiagnostics (RID) and therapy (RIT) is most closely relatedto the present invention.

Bifunctional metal chelating agents for use in antibody conjugate-basedtreatment and diagnostic applications must 1) have functional groupssuitable for conjugation to the antibody, 2) form covalent linkages thatare stable in vivo and which do not destroy-the immunological competenceof the antibody, 3) be relatively nontoxic, and 4) bind and retain theradiometal of interest under physiological conditions.¹⁸⁷⁻¹⁹¹ The lastof these conditions is particularly severe. The potential damage arisingfrom "free" radioisotopes, released from the conjugate, can be veryserious. On the other hand, only nanomole concentrations of isotopes,and hence ligand, are generally required for RID and RIT applications,so that the concerns associated with intrinsic metal and/or free ligandtoxicity are somewhat relaxed.

For the purposes of imaging, an ideal isotope should be readilydetectable by available monitoring techniques and induce a minimalradiation-based toxic response. In practice these and other necessaryrequirements implicate the use of a γ-ray emitter in the 100 to 250 KeVrange, which possesses a short effective half-life (biological and/ornuclear), decays to stable products, and, of course, is readilyavailable under clinical conditions.¹⁷⁸⁻¹⁸⁰ To date, therefore, mostattention has focused on ¹³¹ I (t_(1/2) =193 h), ¹²³ I(t_(1/2) =13 h),^(99m) Tc(t_(1/2) =6.0 h), ⁶⁷ Ga(t_(1/2) =78 h), and ¹¹¹ In(t_(1/2)=67.4 h) which come closest to meeting these criteria.¹⁹² Each of theseenjoys advantages and disadvantages with respect to antibody labelingfor RID. ¹³¹ I and 123I, for instance, are easily conjugated toantibodies via electrophilic aromatic substitution of tyrosineresidues.¹⁹³ The metabolism of ¹³¹ I or ¹²³ I labeled proteins, however,produces free radioactive iodide anion and as a result can lead to afair concentration of radioactivity at sites other than those targetedby the antibody-derived "magic bullet".¹⁹³ The half-lives of both 131Iand 123I are relatively inconvenient for optimal use, being too long andtoo short, respectively, and the fact that ¹³¹ I is also a β emitter.¹⁹²99m Tc, ⁶⁷ Ga and ¹¹¹ In all suffer from the disadvantage that theycannot be bound directly to the antibody in a satisfactory fashion andrequire the use of a bifunctional conjugate. The chemistry of suchsystems is furthest advanced in the case of ^(99m) Tc, and a number ofeffective ligands, are now available for the purpose of ^(99m) Tcadministration.¹⁷⁸⁻¹⁸⁸,194 This radioisotope has a very short half-lifewhich makes it technically very difficult to work with. Both ⁶⁷ Ga andillin have longer half-lives and possess desirable emission energies.Both are "hard" cations with high charge density in their most commontrivalent forms. No suitable ligands exist for either ¹¹¹ In³⁺ or ⁶⁷Ga³⁺ which form stable nonlabile complexes and which might be suitablefor radioimmunological applications. As described elsewhere hereintexaphyrin forms a kinetically and hydrolytically stable complex withIn³⁺. Such a ligand system may be elaborated and serve as the criticalcore of a bifunctional conjugate for use in ¹¹¹ In-based RID.

Many of the same considerations hold true for radioisotope-based therapyas do for radioisotope-based diagnostics: An ideal isotope must also bereadily available under clinical conditions (i.e. from a simpledecay-based generator),¹⁷⁸ possess a reasonable half-life (i.e. on theorder of 6 hours to 4 weeks), and decay to stable products. In addition,the radioisotope must provide good ionizing radiation (i.e. in the 300KeV to 3 MeV range). A number of β emitters, including ¹³¹ I, arecurrently receiving attention as possible candidates for RIT. Among themore promising, are ¹⁸⁶ Re (t_(1/2) =90 h, ⁶⁷ Cu (t_(1/2) =58.5 h), and⁹⁰ Y (t_(1/2) =65 h). Of these, ⁹⁰ Y is currently considered thebest,¹⁹²,197 with an emission energy of 2.28 MeV, it is calculated todeliver roughly 3 to 4 times more energy (dose) to the tumor pernanomole than either ¹⁸⁶ Re or ⁶⁷ Cu. Good immuno-compatible chelandsexist for only ¹⁸⁶ Re and ⁶⁷ Cu, the former may be attached using thesame ligands as were developed for ^(99m) Tc,¹⁹⁴ and the latter via therationally-designed activated porphyrins developed by Prof. Lavallee ofHunter College and the Los Alamos INC-11 team.¹⁹¹ Further benefitsshould be derived from a bifunctional conjugate which is capable offorming stable, nonlabile complexes with ⁹⁰ Y³⁺ (which cannot be donewith porphyrins). The texaphyrin ligand of the present invention notonly forms stable complexes with In³⁺ but also binds Y³⁺ effectively. Atexaphyrin-type bifunctional conjugate may be prepared for use in ¹¹¹In-based RID and in ⁹⁰ Y-based RIT. Both ⁹⁰ Y and ¹¹¹ In couldconceivably be attached to an antibody of choice using a functionalizedtexaphyrin. The Y³⁺ and In³⁺ complexes of texaphyrin are formed rapidly(insertion and oxidation times are less than 3 hours) from themethylene-linked reduced precursor, and are hydrolyrically stable in 1:1methanol-water mixtures (the half-lives for decomplexation and/or liganddecomposition exceed 3 weeks in both cases.

The hydroxy-substituted texaphyrin molecules of the present inventionare especially suited for acting as bifunctional chelating agents inantibody conjugate-based treatment since they have functional groupssuitable for conjugation to the antibody, they form covalent linkagesthat are stable in vivo which do not destroy the immunologicalcompetence of the antibody, they are relatively nontoxic, and they arereadily soluble in a physiological environment. A further advantage ofthese soluble texaphyrins is that many of these would be suitable forfurther functionalization. Treatment of carboxylated texaphyrins withthionyl chloride or p-nitrophenol acetate would generate activated acylspecies suitable for attachment to monoclonal antibodies or otherbiomolecules of interest. Standard in situ coupling methods (e.g.1,1'-carbonyldiimidazole (CDI)²⁰²) could be used to effect theconjugation. The ability to attach and deliver a potent photosensitizerdirectly to a tumor locus could have tremendous potential benefit in thetreatment of neoplastic disorders. In addition, this approach will allowa variety of useful radioisotopes such as ⁹⁰ Y and ¹¹¹ In to be attachedto a monoclonal antibody.

The hydroxy-substituted texaphyrin molecules of the present inventionare also suited for delivering radioactivity to a tumor on their ownsince they chelate radioisotopes and have intrinsic biolocalizationselectivity.

EXAMPLE 10

Magnetio Resonance Imaging Enhancement, Imaging with B2T2 in vivo.

In many respects the key to cancer control lies in early detection anddiagnosis as it does in subsequent therapeutic management. Newtechniques which allow neoplastic tissue to be observed and recognizedat an early stage of development thus have a critical role to play inthe battle against these disorders. One such promising technique ismagnetic resonance imaging (MRI).¹³⁶⁻¹⁴⁰ Although quite new, thisnoninvasive, apparently innocuous method, is now firmly entrenched as adiagnostic tool of prime importance, complementing or, in some cases,supplanting computer assisted X-ray tomography as the method of choicefor solid tumor detection.

The physical basis of current MRI methods has its origin in the factthat in a strong magnetic field the nuclear spins of water protons indifferent tissues relax back to equilibrium at different rates. Whenthese local, tissue-dependent relaxation differences are large, tissuedifferentiation can be effected. Paramagnetic compounds, containing oneor more unpaired spins, enhance the relaxation rates for the waterprotons in which they are dissolved.¹⁴¹ The extent of this enhancementis termed relaxivity. At present, only one paramagnetic MRI contrastagent is in clinical use, the bis(N-methyl-glucamine) salt of Gd(III)diethylenetriaminepentaacetate, (MEG)₂ [Gd(DTpA)(H₂ O)](c.f. structure10)¹⁴⁶⁻¹⁵³ marketed by Berlex Laboratories. This dianionic complexlocalizes selectively in extracellular regions, and is being usedprimarily in the visualization of the capillary lesions associated withcerebral tumors.¹⁴⁶⁻¹⁴⁸

Considerable effort has been devoted to the development of new potentialMRI contrast agents.¹⁵⁶ Most of this work has centered around preparingnew complexes of Gd(III).¹⁵⁶⁻¹⁶⁴,171-172

The emphasis on Gd(III) salts stems from the fact that this cation, with7 unpaired f-electrons, has a higher magnetic moment than otherparamagnetic cations such as Fe(III) and Mn(II).¹³⁹⁻¹⁴⁰ Thus complexesof Gd(III) would be expected to be superior relaxation agents than thosederived from Mn(II) or Fe(III). In addition, both iron and, to a lesserextent, manganese are sequestered and stored very efficiently in humans(and many other organisms) by a variety of specialized metal-bindingsystems.¹⁷³ Moreover both iron and manganese are capable of existing ina range of oxidation states and are known to catalyze a variety ofdeleterious Fenton-type free-radical reactions.¹⁷⁴ Gadolinium(III),which suffers from neither of these deficiencies, thus appears to offermany advantages. As is true for Fe(III) and Mn(II), the aqueous solutionof Gd(III) is too toxic to be used directly for MRI imaging at the 0.01to 1 mM concentrations required for effective enhancement.¹³⁹,140 Hencethe emphasis is on developing new agents which, as is true for DTPA,form hydrolytically stable complexes in vivo with Gd(III) and/or otherparamagnetic cations. A number of such ligands, including the verypromising DOTA¹⁵⁶⁻¹⁶² and EHPG¹⁶³,164 systems, are now known (c.f.reference 140 for an extensive review). In almost all cases, however,reliance is made on the same basic philosophical approach. Specifically,for Gd(III) binding, carboxylates, phenolares, and/or other anionicchelating groups are being used to generate intrinsically labilecomplexes of high thermodynamic stability in the hope that such highthermodynamic stability will translate into a kinetic stability that issufficient for in vivo applications. Little effort is currently beingdevoted to the preparation of nonlabile Gd(III) complexes that would inand of themselves enjoy a high kinetic stability. The problem seems tobe quite simply that such systems are hard to make. For instance, unlikethe transition metal cations which are bound well to porphyrins (asynthetically versatile ligand which is readily subject to modificationand which, at least for [Mn(III)TPPS]¹³⁸, and other water solubleanalogues,¹⁶⁵⁻¹⁶⁹ shows good relaxivity and good tumor localizingproperties), Gd(III) forms only weak and/or hydrolytically unstablecomplexes with porphyrins,^(165c),169,175 although other simplemacrocyclic amine- and imine-derived ligands¹⁷¹,172,176 will supportstable complexes with certain members of the lanthanide series and doshow some promise, as yet unrealized, of acting as supporting chelandsfor Gd(III)-based MRI applications.

According to the present invention nonlabile Gd(III) complexes ofhydroxy-substituted texaphyrins prove to be useful contrast agents forMRI applications. Hydroxy-substituted texaphyrins are capable ofstabilizing complexes with a variety of di- and trivalent cationsincluding Cd²⁺, Hg²⁺, Lu⁺³, Gd⁺³, and La⁺³. Such complexes areparticularly soluble in physiological environments.

Magnetic Resonance Imaging with B2T2 in vivo

The T2B2 gadolinium complex showed low toxicity and good tissueselectivity in magnetic resonance imaging enhancement.

Imaging: Scanning was performed using a circumferential transmit/receivecoil (Medical Advances, Milwaukee, Wis.) in the bore of a 1.5 TeslaSigna scanner (GE Medical Systems, Milwaukee, Wis.). Normal maleSprague-Dawley rats (n=5) weighing from 280-320 grams and rats bearingsubcutaneously implanted methylcholanthrene-induced fibrosarcomas intheir left flanks (n=4) were studied. Tumor size at the time of thestudy ranged from 2.5 to 3.5 cm in widest diameter. The rats wereanesthetized with 90 mg/kg of ketamine (Vetalar, Aveco Corporation, FortDodge, IO) and 10 mg/kg of xylazine (Rompun, Mobay Corporation, Shawnee,Kans.) intraperitoneally. Following the insertion of an intravenouscatheter in the tail vein, each animal was placed in supine (normalrats) or prone (tumor-bearing rats) position in the center of the coil.Coronal and axial T1 weighted images were obtained of each animal usinga spin echo pulse sequence with the following parameters: TR 300 msec,TE 15 msec, slice thickness 5 mm, matrix 128×256, field of view 10 cm, 4excitations and no phase wrap. Next, 17 umol/kg of the Gd(III)texaphyrincomplex dissolved in normal saline was infused at a rate of 0.25 ml/minintravenously and repeat images were obtained at 10-15 minutes postcontrast. One tumor-bearing rat was studied at 6 and 28 hourspost-contrast. All tuning parameters and the rats' positions were keptidentical in the pre and post contrast scans.

Image Analysis: Operator defined regions of interest (ROI) measurementswere made on axial slices of all pre and 10-15 minutes post contraststudies. Regions in which measurements were made included the rightlobes of the livers and the whole kidneys in the normal rats and thewhole tumor in tumor-bearing rats. In addition, large ROI's ofbackground air were measured for standardization purposes. Standardizedsignal intensities (SSI) were calculated as follows: signal intensity(SI) of organ/SI air. An unpaired Student's test was used to compare precontrast and post contrast SSIs.

Toxicity: At 24 hours, there were no deaths in the mice injected i.p.although those receiving the highest dose (312.5 umol/kg) appearedlethargic. Autopsies of two mice from each dosage group revealed someedema and pallor of the liver and kidneys in the two groups receivingthe highest doses (312.5 and 156.3 umol/kg). Autopsies from theremaining groups were normal. At 48 hours, the remaining mice (n=3 ineach dosage group) in the two highest dosage groups died. The animals inthe three lower dosage groups demonstrated no morbidity. There was nomortality or evidence of morbidity in the rats during the month ofobservation after scanning.

Enhancement: Liver SSI increased by 81.7% (p <0.001), kidney by 114.9%(p <0.001) and tumor by 49.7% (p <0.02) from pre to 10-15 minutes postcontrast. There was no significant difference in enhancement between theright and left lobe of the liver and between the two kidneys. Precontrast, tumor parenchyma appeared homogeneous and of an intensitysimilar to adjacent muscle. Post contrast, tumor tissue demonstrated amottled pattern of enhancement and was easily distinguished fromadjacent tissues. The MRI appearance reflected the heterogeneousappearance of the tumor grossly which consists of necrotic tissuesurrounded by viable stroma. In addition, in the one animal studied at 6and 28 hours post contrast, there was visible tumor enhancementthroughout the study period. The pattern of enhancement, however,changed over time, with enhancement starting at the edges of the tumorinitially and including the center by 28 hours.

These results show that the T2B2 gadolinium complex is an hepatic, renaland tumor-specific contrast agent. The agent was found to haverelatively low toxicity in rodents. Intravenous administration resultedin statistically significant hepatic, renal and tumor enhancement inrats within 10-15 minutes with persistence of tumor enhancement for upto 28 hours. The early enhancement of tumor edges may represent contrastlocalization in areas of viable tumor. The later appearance of the tumorprobably was caused by passive diffusion of some of the agent intocentral necrotic areas. It is unclear whether a selective transport orpassive diffusion mechanism is responsible for initial tumor enhancementwith GD(III)texaphyrin and whether intracellular binding toperipheral-type benzodiazepene receptors occurs. The tumor could bedifferentiated from adjacent tissues for up to 28 hours.

The chemical properties of this texaphyrin class of macrocyclic ligandscan be varied by peripheral substitution, which would allow biologicalproperties to be optimized in terms of biodistribution, pharmacokineticsand toxicity.

Magnetic Resonance Imaging of Atheroma.

The gadolinium complex of B2T2[4,5-diethyl-10,23-dimethyl-9,24-bis(3-hydroxypropyl)-16,17-(3-hydroxypropyloxy)-13,20,25,26,27-pentaazapentacyclo[20.2.1.1³,6.1⁸,11.0¹⁴,19 ]heptacosa-1,3,5,7,9,11(27),12,14(19),15,17,20,22(25),23-tridecaene] shows accumulation in humancadaveric aorta. Two aortas obtained from autopsies were examined usingmagnetic resonance imaging before and after incubation in vitro for 15minutes with the gadolinium complex of B2T2. Selective labeling of theendothelial cell surface and atheromas plaque relative to surroundingtissue was observed. These data indicate that the Gd(III)B2T2 complexhas utility in the non-invasive imaging of atheroma.

Magnetic Resonance Imaging of the Upper GI Tract. The gadolinium complexof B2T2[4,5-diethyl-10,23-dimethyl-9,24-bis(3-hydroxypropyl)-16,17-(3-hydroxypropyloxy)-13,20,25,26,27-pentaazapentacyclo[20.2.1.1³,6.1⁸,11.0¹⁴,19 ]heptacosa-1,3,5,7,9,11(27),12,14(19),15,17,20,22(25),23-tridecaene] shows accumulation in the upper GItract, especially the stomach, as determined by magnetic resonanceimaging.

EXAMPLE 11

Photodynamic Therapy, In vitro and In vivo Experiments

In vitro data and experiments. The lanthanum complex of B2T2[4,5-diethyl-10,23-dimethyl-9,24-bis(3-hydroxypropyl)-16,17-(3-hydroxypropyloxy)-13,20,25,26,27-pentaazapentacyclo[20.2.1.1³,6.1⁸,11.0¹⁴,19 ]heptacosa-1,3,5,7,9,11(27),12,14(19),15,17,20,22(25),23-tridecaene] (LaB2T2) was used at concentrationsof 5.0, 1.0 or 0.1 micromolar in tissue culture medium. The murinemammary carcinoma cell line designated EMT-6 was cultured in mediumcontaining LaB2T2 for 1 hour or 3 hours in the dark. Experimentalcultures were irradiated with 10 Joules/cm² using an arc lamp with a 750nanometer band pass filter. Cell survival was measured using a cellcloning assay. There was no dark toxicity indicating that LaB2T2 had nodirect toxicity to the cells. Cultures which were irradiated with thevisible red light showed viabilities of 3%, 50% and 100% forconcentrations of LaB2T2 of 5.0, 1.0 and 0.1 micromolar respectively.The results were similar for 1 and 3 hour incubation periods. Theresults established that LaB2T2 was phototoxic to these tumor cells invitro.

In vivo experiments. Murine adenocarcinoma cells were inoculated intoboth flanks of Balb/c mice. Four days later, palpable tumor masses werepresent on both flanks of the mice. Ten mg/kg of lutetium B2T2 (LuB2T2)in aqueous solution was injected IV. Seven hours later, one tumor masswas irradiated with 500 Joules of Argon laser light at 746 nanometers.The unirradiated tumor served as a control. Animals were monitored dailyand tumor measurements were made using calipers. Following a singletreatment, 65% cell kill was estimated based on the reduction in size ofthe treated tumors. No phototoxicity of skin or normal tissuessurrounding the tumors was observed indicating relatively selectiveuptake of the LuB2T2 in the tumors. This experiment established the invivo photodynamic activity of LuB2T2 in vivo.

The hydroxy-substituted texaphyrins can be conjugated to biologicalmolecules, especially proteins of molecular weight greater than about20,000 daltons, e.g. albumin and gamma globulin, in order to slow theirclearance by the kidneys. A prolonged presence of these complexes intissue may be desirable for photoirradiation purposes. The conjugationwould be accomplished as described in Example 9 for antibody conjugates.

EXAMPLE 12

Hydroxy-Substituted Texaphyrins in Magnetic Resonance Imaging followedby Photodynamic Therapy for Tumor Destruction

This example describes a use of the present invention of hydroxysubstituted texaphyrins in the destruction of tumor tissue. A detectablemetal complex of a water soluble hydroxy-substituted aromaticpentadentate expanded porphyrin analog retaining lipophilicity, saidcomplex exhibiting selective biolocalization in benign or malignanttumor cells relative to surrounding tissue is administered as a firstagent to a host harboring benign or malignant tumor cells. Localizationsites in the host are determined by reference to the detectable metal. Awater soluble hydroxy-substituted aromatic pentadentate expandedporphyrin analog-detectable-metal complex retaining lipophilicity andhaving essentially identical biolocalization property and exhibiting theability to generate singlet oxygen upon exposure to light will beadministered as a second agent. The second agent is photoirradiated inproximity to the benign or malignant tumor cells, as is using fiberoptics, to cause tumor tissue destruction from the singlet oxygenproduced. The water soluble hydroxy-substituted aromatic pentadentateexpanded porphyrin analog retaining lipophilicity is ahydroxy-substituted texaphyrin although one skilled in the art can seefrom the foregoing that substituted sapphyrins, pentaphyrins or othermacrocyclic ligands capable of chelating a metal, soluble in aqueousfluids and localizing in a lipid rich environment may be of particularvalue. The detectable metal in the first agent is a paramagnetic metal,preferably Gd(III) or a gamma emitting metal. The localization sites aredetermined using MRI when a paramagnetic metal is used and gamma bodyscanning when a gamma emitting metal is used. The detectable metal inthe second agent is a diamagnetic metal, preferably La(III), Lu(III) orIn(III). Texaphyrin-metal complexes will be chosen which themselves showa high intrinsic biolocalization selectivity for tumors or neoplastictissues. For example, the B2T2 Gd(III) complex demonstrates in vivoaffinity for tissue high in lipid content, atheroma, the liver, kidneysand tumors. When appropriately followed by fiber optic photodynamictherapy, cells in the atheroma or tumor can be deactivated.

The hydroxy substituted diamagnetic texaphyrin complexes are goodcandidates for such biomedical photosensitizers. They are easilyavailable, have low intrinsic cytotoxicity, long wavelength absorption,generate singlet oxygen, are soluble in physiological environments, havethe ability to be conjugated to site specific transport molecules, havequick elimination, are stable and are easily subject to syntheticmodification.

Literature citations in the following list are incorporated by referenceherein for the reasons cited.

REFERENCES

1. "The Porphyrins"; Dolphin, D., Ed.; Academic Press: New York,1978-1979; Vols. I-VII.

2 "Superphthalocyanine" a pentaaza aromatic phthalocyanine-like systemwas prepared by a uranyl-medicated condensation; it is not obtainable asthe free-base or in other metal-containing forms: (a) Day, V. W.; Marks,T. J.; Wachter, W. A. J. Am. Chem. Soc. 1975, 97, 4519-4527. (b) Marks,T. J.; Stojakovic, D. R. J. Am. Chem. Soc. 1978, 100, 1695-1705. (c)Cuellar, E. A.; Marks, T. J. Inorg. Chem. 1981, 20, 3766-3770.

3. Bauer, V. J.; Clive, D. R.; Dolphin, D.; Paine, J. B. III; Harris, F.L.; King, M. M.; Loder, J.; Wang, S. -W. C.; Woodward, R. B. J. Am.Chem. Soc. 1983, 105, 6429-6436. To date only tetracoordinated metalcomplexes have been prepared from these potentially pentadentateligands.

4. For an example of a porphyrin-like system with a smaller centralcavity, see: (a) Vogel, E; Kocher, M.; Schmickler, H.; Lex, J. Angew.Chem. 1986, 98, 262-263; Angew. Chem., Int. Ed. Engl. 1986, 25, 257-258.(b) Vogel, E.; Balci, M.; Pramod, K.; Koch, P.; Lex, J.; Ermer, 0.Angew. Chem. 1987, 26, 928-931; Angew. Chem., Int. Ed. Engl. 1987, 26,928-931.

5. Mertes et al. have recently characterized a five-coordinate coppercomplex of an elegant (but nonaromatic) porphyrin-like "accordion"ligand derived from dipyrromethines: (a) Acholla, F. V.;.Mertes, K. B.Tetrahedron Lett. 1984, 3269-3270. (b) Acholla, F. V.; Takusagawa, F.;Mertes, K. B. J. Am. Chem. Soc. 1985, 6902-6908. Four-coordinate coppercomplexes of other nonaromatic pyrrole-containing macrocycles have alsobeen prepared recently: Adams, H.; Bailey, N. A.; Fenton, D. A.; Moss,S.; Rodriguez de Barbarin, C. O.; Jones, G. J. Chem. Soc., Dalton Trans.1986, 693-699; Fenton, D. E.; Moody, R. J. Chem. Soc., Dalton Trans.1987, 219-220.

6. Broadhurst, M. J.; Grigg, R; Johnson, A. W. J. Chem. Soc., PerkinTrans. 1 1972, 2111-2116.

7. (a) Broadhurst, M. J.; Grigg, R.; Johnson, A. W. J. Chem. Soc., Chem.Commun. 1969, 23-24. Broadhurst, M. J.; Grigg, R.; Johnson, A. W. J.Chem. Soc., Chem. Commun. 1969, 1480-1482. Broadhurst, M. J.; Grigg, R.;Johnson, A. W. J. Chem. Soc., Chem. Commmun. 1970, 807-809.

8. (a) Berger, R. A.; LeGoff, E. Tetrahedron Lett. 1978, 4225-4228. (b)LeGoff. E.; Weaver, O. G. J. Org. Chem. 1987, 710-711.

9. (a) Rexhausen, H.; Gossauer, A. J. Chem. Soc. Chem. Commun. 1983,275. (b) Gossauer, A. Bull. Soc. Chim. Belg. 1983, 92, 793-795.

10. Gosmann, M.; Franck, B. Angew. Chem. 1986, 98, 1107-1108; Angew.Chem., Int. Ed. Engl. 1986, 25, 1100-1101.

11. The systematic name for compounds 2 is4,5,9,24-tetraethyl-10,23-dimethyl-13,20,25,26,27-pentaazapentacyclo[20.2.1.1³,6.1⁸,11.0¹⁴,19]heptacosa-1,3,5,7,9,11(27),12,14,16,18,20,22(25),23-tridecaene.

12. Nonaromatic planar pentadentate pyridine-derived ligands are known.See, for instance: (a) Curtis, N. F. In Coordination Chemistry ofMacrocyclic Compounds; Melson, G. A., Ed.; Plenum: New York, 1979;Chapter 4. (b) Nelson, S. M. Pure Appl. Chem. 1980, 52, 2461-2476. (c)Ansell, C. W. G.; Lewis, J.; Raithby, P. R.; Ramsden, J. N.; Schroder,M. J. Chem. Soc., Chem. Commun. 1982, 546-547. (d) Lewis, J.;O'Donoghue, T. D.; Raithby, P. R. J. Chem. Soc., Dalton Trans. 1980,1383-1389. (e) Constable, E. C.; Chung, L.-Y.; Lewis, J.; Raithby, P. R.J. Chem. Soc., Chem. Commun. 1986, 1719-1720. (f) Constable, E. C.;Holmes, J. M.; McQueen, R. C. S. J. Chem. Soc., Dalton Trans. 1987, 5-8.

13. (a) Sessler, J. L.; Johnson, M. R.; Lynch, V. J. Org. Chem. 1987,52, 4394-4397. (b) Sessler, J. L.; Murai, T.; Lynch, V.; Cyr, M. J. Am.Chem. Soc. 1988, 110, 5586-5588.

14. Sessler, J. L.; Johnson, M. R.; Lynch, V.; Murai, T. J. Coord.Chem., 1988, 18, 99-104

15. Satisfactory spectroscopic, mass spectrometric, and/or analyticaldata were obtained for all new compounds.

16. OEP=octaethylporphyrin and TPP=tetraphenylporphyrin; the prefixes H₂and Cd refer to the free-base and cadmium(II) forms, respectively;pyr=pyridine.

17. (a) Scheer, H.; Katz, J. J. In Porphyrins and Metalloporphyrins;Smith, K., Ed.; Elsevier: Amsterdam, 1975; Chapter 10. (b) Janson, T.R.; Katz, J. J.; in ref. 1, Vol. IV, Chapter 1.

18. Gouterman, M., in ref. 1, Vol. III, Chapter 1.

19. Becker, R. S.; Allison, J. B. J. Phys. Chem. 1963, 67, 2669.

20. Texaphyrin 1_(c).NO₃ was crystallized from CHCl₃ -hexanes in atriclinic space group.

21. Hoard, J. L., In Porphyrins and Metalloporphyrins; Smith, K., Ed.;Elsevier: Amsterdam, 1975; Chapter 8.

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38. For recent examples of bipyridine-derived systems and relatedpentadentate ligands, see: (a) Ansell, C. W. G.; Lewis, J.; Raithby, P.R.; Ramsden, J. N.; Schroder, M. J. Chem. Soc., Chem. Commun., 1982,546-547. (b) Lewis, J.; O'Donoghue, T. D.; Raithby, P. R. J. Chem. Soc.,Dalton Trans., 1980, 1383-1389. (c) Constable, E. C.; Chung, L.-Y.;Lewis, J.; Raithby, P. R. J. Chem. Soc., Chem. Commun., 1986, 1719-1720.(d) Constable, E. C.; Holmes, J. M.; McQueen, R. C. S. J. Chem. Soc.,Dalton Trans., 1987, 5-8.

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What is claimed is:
 1. A method for synthesizing an aromatic texaphyrinmetal complex having at least one hydroxy substituent, the methodcomprising:mixing a nonaromatic texaphyrin having at least one hydroxysubstituent, a metal cation, a Br.o slashed.nsted base and an oxidantselected from the group consisting of air, oxygen, platinum oxide and2,3-dichloro-1,4-benzoquinone, in an organic solvent that dissolyes thenonaromatic texaphyrin; and stirring at ambient temperature or heatingthe mixture at reflux for at least two hours to form an aromatictexaphyrin metal complex having at least one hydroxy substituent.
 2. Themethod of claim 1 wherein the nonaromatic texaphrin having at least onehydroxy substituent is produced by condensation of a diformyltripyrrolehaving structure A; and an ortho-phenylenediamine having structure B:##STR12## where Rhd 1, R₂, R₃, R₄, and R₅ are independently H, OH,alkyl, oxyalkyl, hydroxyalkyl, oxyhydroxyalkyl, monosaccharide,carboxyalky or carboxyamidealkyl; where at least one of R₁, R₂, R₃, R₄,and R₅ is oxyhydroxyalky, monosaccharide, oxyalkyl, carboxyalkyl,carboxyamidealkyl or hydroxyalkyl; and the molecular weight of any oneof R₁, R₂, R₃, R₄, or R₅ is less than or equal to about 1000 daltons. 3.The method of claim 2 wherein the diformyltripyrrole is2,5-bis[(5-formyl-3-hydroxyalkyl-4-alkylpyrrol-2-yl)methyl]-3,4-dialkylpyrroleand the ortho-phenylenediamine is1,2-diamino-4,5-bis(oxyhydroxyalkyl)benzene.
 4. The method of claim 2wherein the diformyltripyrrole is2,5-bis[(5-formyl-3-hydroxyalkyl-4-alkylpyrrol-2-yl)methyl]-3,4-dialkylpyrrole.5. The method of claim 2 wherein the orthophenylenediamine is1,2-diamino-4,5-bis(oxyhydroxyalkyl)benzene.
 6. The method of claim 5wherein the orthophenylenediamine is1,2-diamino-4,5-bis[(2,3-hydroxypropyl)oxy]benzene.
 7. The method ofclaim 5 wherein the orthobenylenediamine is1,2-diamino-4,5-bis[(2,3-dihydroxypropyl)oxy]benzene.
 8. The method ofclaim 4 wherein the diformyltripyrrole is2,5-bis[(5-formyl-3-hydroxypropyl-4-methylpyrrol-2-yl)methyl]-3,4-diethylpyrrole.9. The method of claim 4 wherein the diformyltripyrrole is2,5-bis[(3-ethyl-5-formyl-4-methylpyrrol-2-yl)methyl]-3,4-diethylpyrrole.10. The method of claim 1 wherein the Br.o slashed.nsted base istriethylamine.
 11. The method of claim 1 wherein the oxidant is air andthe air is saturating the organic solvent.
 12. The method of claim 1wherein the stirring or heating at reflux step is stirring or heating atreflux the mixture for at least 24 hours.
 13. The method of claim 1wherein the organic solvent comprises methanol and chloroform.
 14. Themethod of claim 1 wherein the organic solvent comprises methanol andbenzene.
 15. The method of claim 2 whereR₁, R₂, R₃, and R₄ areindependently hydrogen or C_(n) H_(2n+1) where n is a positive integerfrom 1 to about 10; and R₅ is hydroxyl, hydroxyalkyl, oxyhydroxyalkyl,carboxyalkyl or carboxyamidealkyl.
 16. The method of claim 2 whereR₁,R₂, R₃, and R₄ are independently hydrogen, hydroxyl, alkyl,hydroxyalkyl, oxyhydroxyalkyl, carboxyalkyl or carboxyamidealkyl, and R₅is H or C_(n) H_(2n+1) where at least one of R₁, R₂, R₃, and R₄ ishydroxyl, hydroxyalkyl, carboxyaikyl, carboxyamidealkyl oroxyhydroxyalkyl; and n is a positive integer from 1 to
 10. 17. Themethod of claim 2 whereR₁, R₂, and R₃ are CH₂ CH₃, R₄ is CH₃, and R₅ isOCH₂ CH₂ CH₂ OH.
 18. The method of claim 2 whereR₁ is (CH₂)₂ CH₂ OH, R₂and R₃ are CH₂ CH₃, and R₄ and R₅ are CH₃.
 19. The method of claim 2whereR₁ is (CH₂)₂ CH₂ OH, R₂ and R₃ are CH₂ CH₃, R₄ is CH₃, and R₅ isOCH₂ CH₂ CH₂ OH.
 20. The method of claim 2 whereR₁, R₂, and R₃ are CH₂CH₃, R₄ is CH₃, and R₅ is OCH₂ CHOHCH₂ OH.
 21. The method of claim 2whereR₁ is (CH₂)₂ CH₂ OH, R₂ and R₃ are CH₂ CH₃, R₄ is CH₃, and R₅ isOCH₂ CHOHCH₂ OH.
 22. The method of claim 2 whereR₁ is (CH₂)₂ CH₂ OH, R₂is CH₂ CH₂ OH, R₃ is CH₂ CH₃, R₄ is CH₃, and R₅ is OCH₂ CHOHCH₂ OH. 23.The method of claim 2 wherein the oxyhydroxyalkyl is C.sub.(n-x)H.sub.((2n+1)-2x) O_(x) O_(y) or OC.sub.(n-x) H.sub.((2n+1)-2x) O_(x)O_(y) wheren is a positive integer from 1 to 10; x is zero or a positiveinteger less than or equal to n; and y is zero or a positive integerless than or equal to ((2n+1)-2x).
 24. The method of claim 2 wherein theoxyhydroxyalkyl or monosaccharide is C_(n) H.sub.(2n+1)-q) O_(y) R^(a)_(q), OC_(n) H.sub.((2n+1)-q) O_(y) R^(a) _(q) or (CH₂)_(n) CO₂ R^(a)wheren is a positive integer from 1 to 10, y is zero or a posimiveinteger less than ((2n+1)-q), q is zero or a positive integer less thanor equal to 2n+1, R^(a) is independently H, alkyl, hydroxyalkyl,monosaccharide, C.sub.(m-w) H.sub.((2m+1)-2w) O_(w) O_(z), O₂CC.sub.(m-w) H.sub.((2m+1)-2w) O_(w) O_(z) or N(R)OCC.sub.(m-w)H.sub.((2m+1)-2w) O_(w) O_(z), wherem is a positive integer from 1 to10, w is zero or a positive integer less than or equal to m, z is zeroor a positive integer less than or equal to ((2m+1)-2w), R is H, alkylhydroxyalkyl, or C_(m) H.sub.((2m+1)-r) O_(z) R^(b) _(r) wherem is apositive integer from 1 to 10, z is zero or a positive integer less than((2m+1)-r), r is zero or a positive integer less than or equal to 2m+1,and R^(b) is independently H, alkyl, hydroxyalkyl, or monosaccharide.25. The method of claim 2 wherein the carboxyamidealkyl is (CH₂)_(n)CONHR^(a), O(CH₂)_(n) CONHR^(a), (CH₂)_(n) CON(R^(a))₂, or O(CH₂)_(n)CON(R^(a))₂ wheren is a positive integer from 1 to 10, R^(a) isindependently H, alkyl, hydroxyalkyl, monosaccharide, C.sub.(m-w)H.sub.((2m+1)-2w) O_(w) O_(z), O₂ CC.sub.(m-w) H.sub.((2m+1) O_(w) O_(z)or N(R)OCC.sub.(m-w) H.sub.((2m+1)-2w) O_(w) O_(z), where m is apositive integer from 1 to 10, w is zero or a positive integer less thanor equal to m, z is zero or a positive integer less than or equal to((2m+1)-2w), R is H alkyl, hydroxyalkyl, or C_(m) H.sub.((2m+1)-r) O_(z)R^(b) _(r) wherem is a positive integer from 1 to 10, z is zero or apositive integer less than ((2m+1)-r), r is zero or a positive integerless than or equal to 2m+1, and R^(b) is independently H, alkyl,hydroxyalkyl, or monosaccharide.
 26. The method of claim 2 wherein thecarboxyalkyl is C_(n) H.sub.((2n+1)-q) O_(y) R^(c) _(q) or OC_(n)H.sub.((2n+1)-q) O_(y) R^(c) _(q) wheren is a positive integer from 1 to10; y is zero or a positive integer less than ((2n+1)-q) , q is zero ora positive integer less than or equal to 2n+1, R^(c) is (CH₂)_(n) CO₂R^(d), (CH₂)_(n) CONHR^(d) or (CH₂)_(n) CON(R^(d))₂ wheren is a positiveinteger from 1 to 10; R^(d) is independently H, alkyl, hydroxyalkyl,monosaccharide, C.sub.(m-w) H.sub.((2m+1)-2w) O_(w) O_(z), O₂CC.sub.(m-w) H.sub.((2m+1)-2w) O_(w) O_(z) or N(R)OCC.sub.(m-w)H.sub.((2m+1)-2w) O_(w) O_(z), where m is a positive integer from 1 to10, w is zero or a mositive integer less than or equal to m, z is zeroor a positive integer less than or equal to ((2m+1)-2w), R is H, alkyl,hydroxyalkyl, or C_(m) H.sub.((2m+1)-r) O_(z) R^(b) _(r) wherem is apositive integer from 1 to 10, z is zero or a positive integer less than((2m+1)-r), r is zero or a Dositive integer less than or equal to 2m+1,and R^(b) is independently H, alkyl, hydroxyalkyl, or monosaccharide.27. A method for synthesizing a metal complex of4,5-diethyl-10,23-dimethyl-9,24-bis(3-hydroxypropyl)-16,17-(3-hydroxypropyloxy)-13,20,25,26,27-pentaazapentacyclo[20.2.1.1³,6.1⁸,11.0¹⁴,19]heptacosa-1,3,5,7,9,11(27),12,14(19),15,17,20,22(25),23-tridecaene, themethod comprising:mixing4,5-diethyl-10,23-dimethyl-9,24-bis(3-hydroxypropyl)-16,17-bis(3-hydroxypropyloxy)-13,20,25,26,27-pentaazapentacyclo[20.2.1.1³,6.1⁸,11.0.sup.14,19]heptacosa-3,5,8,10,12,14(19),15,17,20, 22,24-undecaene, a trivalentmetal cation selected from the group consisting of Mn⁺³, Co⁺³, Ni⁺³,Fe⁺³, Ho⁺³, Ce⁺³, Y⁺³, In⁺³, Pr⁺³, Nd⁺³, Sm⁺³, Eu⁺³, Gd³, Tb⁺³, Dy⁺³,Er³, Tm⁺³, Yb⁺³, Lu⁺³, La⁺³, and U⁺³, a Br.o slashed.nsted base and anoxidant selected from the group consisting of air, oxygen, platinumoxide and 2,3-dichloro-5,6-dicyano-1,4-benzoquinone. in an organicsolvent that dissolves4,5-diethyl-10,23-dimethyl-9,24-bis(3-hydroxypropyl)-16,17-bis(3-hydroxypropyloxy)-13,20,25,26,27-pentaaza-pentacyclo[20.2.1.1³,6.1⁸,11.0.sup.14,19]heptacosa-3,5,8,10,12,14(19),15,17,20,22,24-undecaene; and stirring atambient temperature or heating the mixture at reflux for at least twohours to form a metal complex of 4,5-diethyl-10,23-dimethyl-9,24-bis(3-hydroxypropyl)-16,17-(3-hydroxypropyloxy)-13,20,25,26,27-pentaazapentacyclo[20.2.1.1³,6.1⁸,110¹⁴,19 ]heptacosa-1,3,5,7,9,11(27),12,14(19),15,17,20,22(25),23-tridecaene.
 28. The method of claim 27 wherein theBr.o slashed.nsted base is triethylamine.
 29. The method of claim 27wherein the oxidant is air and the air is saturating the organicsolvent.
 30. The method of claim 27 wherein the stirring or heating atreflux step is stirring or heating at reflux the mixture for at least 24hours.
 31. The method of claim 27 wherein the organic solvent comprisesmethanol and chloroform.
 32. The method of claim 27 wherein the organicsolvent comprises methanol and benzene.
 33. The method of claim 27wherein the organic solvent comprises methanol and dimethylformamide.34. The method of claim 1 wherein the metal cation is a trivalent cationselected from the group consisting of Mn⁺³, Co⁺³, Ni⁺³, Fe⁺³, Ho⁺³,Ce⁺³, Y⁺³, In⁺³, Pr⁺³, Nd⁺³, Sm⁺³, Eu⁺³, Gd⁺³, Tb⁺³, Dy⁺³, Er⁺³, Tm⁺³,Yb⁺³, Lu⁺³, La⁺³, and U⁺³.
 35. The method of claim 1 wherein the metalcation is a divalent cation selected from the group consisting of Ca⁺²,Mn⁺², Co⁺², Ni⁺², Zn⁺², Cd⁺² , Hg⁺², Sm⁺², and UO.sub.⁺².
 36. The methodof claim 1 wherein the organic solvent comprises methanol.
 37. Themethod of claim 27 wherein the organic solvent comprises methanol. 38.The method of claim 2 where R₁ is hydroxyalkyl; R₂, R₃ and R₄ are alkyl;and R₅ is oxyalkyl.
 39. The method of claim 38 where the hydroxyalkyl ofR₁ is CH₂ CH₂ CH₂ OH, the alkyl of R₂ and R₃ is CH₂ CH₃, the alkyl of R₄is CH₃, and the oxyalkyl of R₅ is O(CH₂ CH₂ O)₂ CH₂ CH₂ OR', where R' isH or CH₃.
 40. The method of claim 39 where R' is CH₃.
 41. The method ofclaim 40 where the metal cation is Lu (III) or Gd (III).
 42. A methodfor synthesizing a metal complex of4,5-diethyl-10,23-dimethyl-9,24-bis(3-hydroxypropyl)-16,17-bis[2-[2-(2-methoxyethoxy)ethoxy]ethoxy]-13,20,25,26,27-pentaazapentacyclo[20.2.1.1³,6.1⁸,11.0¹⁴,19]heptacosa-1,3,5,7,9,11(27),12,14(19),15,17,20,22(25), 23-tridecaene,the method comprising:mixing4,5-diethyl-10,23-dimethyl-9,24-bis(3-hydroxypropyl)16,17-bis[2-[2-(2-methoxyethoxy)ethoxy]ethoxy]-13,20,25,26,27-pentaazapentacyclo[20.2.1.1³,6.18,11.0¹⁴,19]heptacosa-3,5,8,10,12,14,16,18,20,22,24-undecaene, a metal cation, aBr.o slashed.nsted base and an oxidant selected from the groupconsisting of air, oxygen, platinum oxide and2,3-dichloro-5,6-dicyano-1,4-benzoquinone, in an organic solvent thatdissolves4,5-diethyl-10,23-dimethyl-9,24-bis(3-hydroxypropyl)-16,17-bis[2-[2-(2-methoxyethoxy)ethoxy]ethoxy]-13,20,25,26,27-pentaazapentacyclo[20.2.1.1³,6.18,11.0¹⁴,19] heptacosa-3,5,8,10,12,14,16,18,20,22,24-undecaene; and stirring atambient temperature or heating the mixture at reflux for at least twohours to form a metal complex of 4,5-diethyl-10,23-dimethyl-9,24-bis(3-hydroxypropyl)-16,17-bis[2-[2-(2-methoxyethoxy)ethoxy]ethoxy]-13,20,25,26,27-pentaazapentacyclo[20.2.1.1³,6.1⁸,11.0¹⁴,19 ]heptacosa-1,3,5,7,9,11(27),12,14 (19),15,17,20,22(25),2 3 -tridecaene.
 43. The method of claim 42 wherein theBr.o slashed.nsted base triethylamine.
 44. The method of claim 42wherein the oxidant is air and the air is saturating the organicsolvent.
 45. The method of claim 42 wherein the organic solvent consistsof methanol and chloroform, methanol and benzene, methanol anddimethylformamide, or methanol.
 46. The method of claim 42 wherein themetal cation is a trivalent cation selected from the group consisting ofMn⁺³, Co⁺³, Ni⁺³, Fe⁺³, Ho⁺³, Ce⁺³, Y⁺³, In⁺³, Pr⁺³, Nd⁺³, Sm⁺³, Eu⁺³,Gd⁺³, Tb⁺³, Dy⁺³, Er⁺³, Tm⁺³, Yb⁺³, Lu⁺³, La⁺³, and U⁺³ or a divalentcation selected from the group consisting of Ca⁺², Mn⁺², Co⁺², Ni⁺²,Zn⁺², Cd⁺², Hg⁺², Sm⁺² and UO₂ ⁺².